The human YAE1-ORAOV1 complex of the cytosolic iron-sulfur protein assembly machinery binds a [4Fe-4S] cluster

Abstract Iron-sulfur (Fe-S) clusters are among the most versatile cofactors in biology. Although Fe-S clusters formation can be achieved spontaneously in vitro with inorganic iron and sulfur sources, the in vivo behaviour is more complex and requires the so-called Fe-S biogenesis machineries. In the cytosol, the biogenesis of Fe-S proteins is assisted by the cytosolic Fe-S protein assembly machinery, which comprises at least thirteen known proteins, among which there are human ORAOV1 and YAE1. A hetero-complex formed by the two latter proteins facilitates Fe-S cluster insertion in the human ABC protein ABCE1 within a chain of binding events that are still not well understood. In the present work, ORAOV1 and the YAE1-ORAOV1 complex were produced and their structural and cluster binding properties spectroscopically investigated. It resulted that both ORAOV1 and the YAE1-ORAOV1 complex are characterized by well-structured, α -helical regions and by unstructured, flexible regions, and are both able to bind a [4Fe-4S]2+ cluster. Bioinformatics and site-directed mutagenesis studies indicated that the [4Fe-4S] cluster in ORAOV1 is bound by a conserved cluster binding motif, while YAE1, which does not have a metal-binding consensus motif, is not essential for the [4Fe-4S]2+ cluster binding in the YAE1-ORAOV1 hetero-complex. Overall, these results support a model that the YAE1-ORAOV1 complex might actively participate in the Fe-S cluster insertion into ABCE1 thanks to the [4Fe-4S]2+ cluster binding properties of ORAOV1

Fragment-based approach to identify IDO1 inhibitor building blocks

Abstract 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. In this paper, we report a fragment-based screening campaign that combines Water-LOGSY NMR experiments and microscale thermophoresis approach to identify fragments that may be helpful for the development of novel IDO1 inhibitors as therapeutic agents in immune-oncology disorders

Potential of Energy Saving of Propane Heat Pump as replacement of gas boilers with low and high temperature emitters

This work deals with the analysis of the energy performance and the environmental impact of a Heating, Ventilation and Air Conditioning (HVAC) system based on an innovative Air-to-Water electrical Heat Pump (AWHP) using propane (R290) as the refrigerant. A building of the University of Bologna located in Forlì (North of Italy) is considered for replacing a condensing gas boiler and a conventional chiller with an AWHP using R290. To evaluate the efficiency of the existing heating system and the potential savings linked to the adoption of the propane AWHP, the building energy model was created and calibrated by collecting monthly thermal and electrical consumptions as a function of the actual climate data. In this paper, the main features of the R290-based AWHP are described in detail by emphasising the device performance as a function of the operating conditions (i.e., air and water temperature and speed of the scroll compressor). A series of scenarios have been studied to evaluate the energy performance of the propane AWHP with respect to the reference scenario under various operating conditions. The results show that while the total primary energy demand increases adopting the propane AWHP with respect to the case of a gas boiler, the non-renewable primary energy fraction decreases significantly, with a dramatic increase in the renewable quote. From an economic point of view, lower annual costs are obtained by adopting a propane AWHP coupled to fan coils, mainly when the electrical heat pump is used in a thermally insulated building in which a photovoltaic system is installed

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. Additionally, an N-terminal fragment of human/mouse ZIP 13 zinc transporter (MPGCPCPGCGMACPR-NH2, later called ZIP13+C), has been chosen for the thermodynamic stability comparison studies. The relative ZIP13+C stability has been shown using several Zn(2+) complexes with artificially arranged multi-cysteine sequences. An interesting coordination mode has been proposed for the IRT1-Zn(2+) complex, in which imidazoles from two histidines (His-96 and His-116), a cysteine thiolate (Cys-109) and one of a glutamic acid carboxyl group are involved. All data were collected using potentiometric, NMR and mass spectrometric methods

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). On the other hand, the availability of systems of different sizes and stabilities has, over the years, stimulated NMR spectroscopists to exploit iron–sulfur proteins as paradigmatic cases to develop experiments, models, and protocols. Here, the cluster-binding properties of human mitoNEET have been investigated by 1D and 2D 1H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated [Fe2S2]2+/+ proteins. The protocol we have developed in this work conjugates spectroscopic information arising from “classical” paramagnetic NMR with an extended mapping of the signals of residues around the cluster which can be taken, even before the sequence-specific assignment is accomplished, as a fingerprint of the protein region constituting the functional site of the protein. We show how the combined use of 1D NOE experiments, 13C direct-detected experiments, and double- and triple-resonance experiments tailored using R1- and/or R2-based filters significantly reduces the “blind” sphere of the protein around the paramagnetic cluster. This approach provided a detailed description of the unique electronic properties of mitoNEET, which are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.</p

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&ndash;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 synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. The present review provides a structural and molecular overview of the rare diseases associated with the genes encoding for the accessory proteins of the ISC machinery (i.e., GLRX5, ISCA1, ISCA2, IBA57, FDX2, BOLA3, IND1 and NFU1) involved in the assembly and insertion of [4Fe-4S] clusters in mitochondrial proteins. The disease-related missense mutations were mapped on the 3D structures of these accessory proteins or of their protein complexes, and the possible impact that these mutations have on their specific activity/function in the frame of the mitochondrial [4Fe-4S] protein biogenesis is described