Cluster Transfer Research Articles

Experimental evidence for α production following neutron transfer in the C13 + Nb93 system

Spectra of $\ensuremath{\alpha}$ particles were measured for $^{12,13}\mathrm{C} + ^{93}\mathrm{Nb}$ systems at several angles for a bombarding energy of 65 MeV to investigate various reaction mechanisms like direct break up and cluster transfer responsible for $\ensuremath{\alpha}$ production. The $\ensuremath{\alpha}$ spectra were analyzed in detail to determine the effect of $1n$ transfer on an $\ensuremath{\alpha}$-production cross section in particular. After accounting for contributions from direct breakup and Be transfer, there is a clear signature of the $\ensuremath{\alpha}$ particles produced through the break-up process following neutron transfer in the case of a $^{13}\mathrm{C}$ induced reaction. This is an interesting result being reported for the strongly bound projectile.

Inhibition of mitoNEET attenuates LPS-induced inflammation and oxidative stress

MitoNEET (mitochondrial protein containing Asn–Glu–Glu–Thr (NEET) sequence) is a 2Fe–2S cluster-containing integral membrane protein that resides in the mitochondrial outer membrane and participates in a redox-sensitive signaling and Fe–S cluster transfer. Thus, mitoNEET is a key regulator of mitochondrial oxidative capacity and iron homeostasis. Moreover, mitochondrial dysfunction and oxidative stress play critical roles in inflammatory diseases such as sepsis. Increased iron levels mediated by mitochondrial dysfunction lead to oxidative damage and generation of reactive oxygen species (ROS). Increasing evidence suggests that targeting mitoNEET to reverse mitochondrial dysfunction deserves further investigation. However, the role of mitoNEET in inflammatory diseases is unknown. Here, we investigated the mechanism of action and function of mitoNEET during lipopolysaccharide (LPS)-induced inflammatory responses in vitro and in vivo. Levels of mitoNEET protein increased during microbial or LPS-induced sepsis. Pharmacological inhibition of mitoNEET using mitoNEET ligand-1 (NL-1) decreased the levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in animal models of sepsis, as well as LPS-induced inflammatory responses by macrophages in vitro. Inhibition of mitoNEET using NL-1 or mitoNEET shRNA abrogated LPS-induced ROS formation and mitochondrial dysfunction. Furthermore, mitochondrial iron accumulation led to generation of LPS-induced ROS, a process blocked by NL-1 or shRNA. Taken together, these data suggest that mitoNEET could be a key therapeutic molecule that targets mitochondrial dysfunction during inflammatory diseases and sepsis.

Time-lapse imaging of CD63 dynamics at the HIV-1 virological synapse by using agar pads.

Time-lapse imaging provides an uninterrupted observation method that can lead to understanding protein dynamics. We previously developed a technique based on thin agar pads to keep the cells in focus during confocal laser scanning microscope imaging. Using this method, time-lapse imaging was employed to monitor CD63 fused to mCherry at the virological synapse (VS) during viral cluster transfer to acceptor cells of the human immunodeficiency virus 1 (HIV-1).

Tuning the fast generation of luminescent silver nanodots on a surface.

Silver nanodots, predominantly a near-IR emitter, can be instantly generated on a surface by silver cluster transfer. Kinetic trapping of ssDNA molecules on the surface limits the reorganization of the resulting silver nanodots for other silver nanodot emitters. Adjusting the freedom of the adsorbed ssDNA can tune the generation of various silver nanodots on the surface.

Tracing the incorporation of the "ninth sulfur" into the nitrogenase cofactor precursor with selenite and tellurite.

The Mo-nitrogenase catalyzes the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of Mo and homocitrate. Previously, we have identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘9th sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘9th sulfur’ insertion remain elusive. Here, we trace the ‘9th sulfur’ insertion using SeO32− and TeO32− as ‘labeled’ SO32−. Biochemical, EPR and XAS/EXAFS studies suggest a role of the ‘9th sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2−- and Te2−-like species into the L-cluster. DFT calculations further point to a plausible mechanism involving in-situ reduction of SO32− to S2−, thereby suggesting the utility of this reaction to label the catalytically-important belt region for mechanistic investigations of nitrogenase.

Механізми реакції 6Li(10B,9Be)7Be при енергії 51 МеВ. Структура та взаємодія ядер 9Be + 7Be

New experimental data of angular distributions for the 6Li(10B,9Be)7Be reaction were measured at the energy Elab(10B) = 51 MeV for the ground states of nuclei and excited 0.429 - 7.2 MeV states of 7Be. The reaction experimental data were analyzed within coupled-reaction-channels method (CRC) for many types of the nucleon and cluster transfers which spectroscopic amplitudes (factors) in the 9Be and 7Be nuclei were calculated using translation invariant shell model. The Woods - Saxon potential was used for CRC-calculations. The 6Li + 10B potential parameters were deduced before from the analysis of experimental data of 10B ions scattering by 6Li nuclei at the energy Elab(10B) = 51 MeV, and the 9Be + 7Be potential parameters for exit reaction channel were deduced from the CRC calculations fitting to the reaction experimental data. Thus, the information about the 9Be + 7Be optical potential, the basic reaction mechanisms and the 9Be and 7Be nuclei structures were deduced.

Pioglitazone Inhibits Metal Cluster Transfer of mitoNEET by Stabilizing the Labile Fe-N Bond Revealed at Single-Bond Level.

Outer mitochondrial membrane protein mitoNEET (mNT) is a target of the type 2 diabetes drug pioglitazone. It contains a labile Fe2S2(His)1(Cys)3 metal cluster with a single Fe-N(His87) coordinating bond and can transfer its cluster to acceptor proteins. Previous ensemble studies showed that pioglitazone's binding inhibited the transfer by stabilizing the cluster, and histidine 87 may be the key mediator. Here we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to study the unfolding process of mNT dimer in the absence and presence of pioglitazone, which can distinguish the binding effect for different regions of a protein. By developing a two-step strategy using different mNT monomers with respective purification tags, we solve the problem that the classic polyprotein formation disables the mNT to dimerize. As a result, a polyprotein including a stable, naturally noncovalently bound mNT homodimer is obtained, which is required for reliable AFM measurement and pioglitazone binding. Then, the dissociation rate (koff) of the metal cluster was measured, showing a 10-fold decrease upon pioglitazone binding, while the other parts decreased only 3-fold, verifying that pioglitazone mainly stabilizes the cluster. Moreover, when the Fe(III)-N(His87) bond was ruptured, this effect for the remaining Fe2S2(Cys)3 intermediate largely disappeared. Consequently, AFM results revealed that pioglitazone inhibited the metal cluster transfer of mNT by stabilizing the labile Fe(III)-N(His87) bond. In addition, an alternative method to build a natural, noncovalently bound protein dimer or complex for reliable single-molecule measurement was developed.

Пружне й непружне розсіяння іонів 15N ядрами 13С при енергії 84 МеВ

New experimental data of the 15N + 13C elastic and inelastic scattering were obtained at the energy Elab(15N) = 84 MeV. The data were analyzed within the coupled-reaction-channels method. The elastic and inelastic scattering of nuclei 15N + 13С as well as the more important nucleon and cluster transfer reactions were included in the channels-coupling scheme. The WS potential parameters for the 15N + 13С nuclei interactions in ground and excited states as well as deformation parameters of these nuclei were deduced. The contributions of one- and two-step transfers in the 15N + 13C elastic and inelastic scattering were estimated. The results of the 15N + 13С elastic scattering at the energy Elab(15N) = 84 MeV, obtained in this work, were compared with that of the 15N + 12С elastic scattering at the energy Elab(15N) = 81 MeV.

ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.

Downregulation of CISD2 Has Prognostic Value in Non-Small Cell Lung Cancer and Inhibits the Tumorigenesis by Inducing Mitochondrial Dysfunction.

CISD2, a NEET protein that coordinates 2Fe-2S clusters through its CDGSH domain, is critical for normal development and iron homeostasis. CISD2 plays an important role in Fe-S cluster transfer and promotes cancer proliferation. However, its specific role in the development of non-small cell lung cancer (NSCLC) remains unclear. Bioinformatics of pan-cancer analysis from The Cancer Genome Atlas show that CISD2 has an aberrant expression in most types of human cancers. Moreover, CISD2 expression is associated with a higher hazard ratio and exhibits significantly poorer overall survival in lung adenocarcinoma (LUAD), uveal melanoma, head and neck squamous cell carcinoma, brain lower grade glioma, kidney chromophobe, and liver hepatocellular carcinoma. Further investigation revealed that CISD2 is highly expressed in LUAD and LUSC, which is associated with clinical pathological stages. In addition, survival data collected from GSE31210 and GSE13213, two datasets from the NCBI Gene Expression Omnibus, also confirmed that high CISD2 expression is associated with unfavorable survival in patients with LUAD. A cell-based assay indicated that the knockdown of CISD2 inhibited proliferation, invasion, and migration in A549 cells. Additionally, CISD2 knockdown accelerated the accumulation of cellular and mitochondrial reactive oxygen species, destroying the mitochondrial morphology and function. Moreover, CISD2 inhibition activated the iron starvation response, thus, accelerating iron accumulation in A549 cells. Pretreatment with DFO, the iron chelator, blocked mitochondrial dysfunction in CISD2-knockdown cells. Collectively, the present study provides novel insights into the regulatory role of CISD2 in NSCLC and presents a potential target to improve antitumor activity based on oxidative stress.

Mammalian mitochondrial iron-sulfur cluster biogenesis and transfer and related human diseases.

As a cofactor, iron-sulfur (Fe-S) cluster binds to proteins or enzymes that play important roles in various important biological processes, including DNA synthesis and repair, mitochondrial function, gene transcription and translation. In mammals, the core components involved in Fe-S cluster biosynthesis are considered to include the scaffold protein ISCU, cysteine desulfurase NFS1 and its accessory proteins ISD11 and ACP, and frataxin (FXN). Proteins involved in Fe-S cluster transfer have been found to include HSC20/HSPA9, as chaperone system, and Fe-S cluster carriers. The biosynthesis and transfer of Fe-S clusters to Fe-S recipients require fine-tune regulation. Recently, significant progress has been made in the structure and mechanism of mitochondrial Fe-S biosynthesis and transfer. Based on, especially, the development of DNA sequencing technology, bioinformatics, and gene editing technology, diseases caused by mutations of Fe-S cluster-related genes have been revealed in recent years, promoting the rapid development in the field of Fe-S and human health. This review focuses on the function of genes involved in Fe-S cluster biosynthesis and transfer and on the diseases caused by the mutations of the related genes. Finally, some questions we are facing are raised, new hypotheses presented, and the perspectives discussed.

Circular Dichroism to Probe the Synthesis, Transfer, and Stability of Fe-S Clusters.

All Fe-S proteins are characterized by distinctive circular dichroism (CD) features in the visible region of the spectrum due to chiral interaction between the cluster itself and the protein backbone. Therefore, the presence of a CD signal in the visible region relates to the presence of the cluster, whereas the disappearance of the signal refers to cluster breakdown or redox changes. The position of the CD features in the spectrum and the intensity of individual components of the CD signal show great variations among different Fe-S proteins. This feature can provide information on transfer processes between proteins, as well as on possible changes in cluster nuclearity. This method can also be used to detect changes in the chemical nature or spatial organization of cluster ligands that may be concurrent with cluster transfer and associated events.

Cluster transfer reactions with the combined R-matrix and Lagrange-mesh methods

We have recently applied the R-matrix method to transfer reactions in the distorted wave Born approximation (DWBA) framework. In our approach the wave function in the internal region is expanded in terms of Lagrange basis, which provides a fast and efficient way to compute the matrix elements. This paper is a short review of our work on transfer reactions. I discuss applications of our approach by considering the $$^{16}$$ O(d, n) $$^{17}$$ F and $$^{12}$$ C( $$^7$$ Li, t) $$^{16}$$ O reactions, which are specific examples of neutron and $$\alpha $$ transfer, respectively. In particular, I discuss the role of the remnant terms, post-prior form equivalence, peripherality of the reaction and sensitivity of the transfer cross sections to the bound state wave functions. Effects of the remnant terms and of the supersymmetric bound state potentials on the extracted spectroscopic factors are also discussed.

Searching for treasures at sub-barrier energies: the case of 8B and 7Be

Unexpected and challenging experimental results, at below barrier energies for weakly bound nuclei, are briefly reviewed in this article. The emphasis will be on our recent breakup results for 8B+208Pb at deep sub-barrier energies, indicating a dominance of direct mechanisms at this low energy regime. We will also present, a preliminary analysis of the 4He and 3He - particle production events for the 7Be + 208Pb reaction. These data were collected at the same experiment and at similar deep sub-barrier energies, exhibiting large yields compatible with cluster transfer processes. To confirm these results a new experiment to be performed at TriSol is planned, as soon as the upgrading of this facility will be completed.

Methods to Investigate the Kinetic Profile of Cysteine Desulfurases.

Biological iron-sulfur (Fe-S) clusters are essential protein prosthetic groups that promote a range of biochemical reactions. In vivo, these clusters are synthesized by specialized protein machineries involved in sulfur mobilization, cluster assembly, and cluster transfer to their target proteins. Cysteine desulfurases initiate the first step of sulfur activation and mobilization in cluster biosynthetic pathways. The reaction catalyzed by these enzymes involves the abstraction of sulfur from the amino acid L-cysteine, with concomitant formation of alanine. The presence and availability of a sulfur acceptor modulate the sulfurtransferase activity of this class of enzymes by altering their reaction profile and catalytic turnover rate. Herein, we describe two methods used to probe the reaction profile of cysteine desulfurases through quantification of alanine and sulfide production in these reactions.

Механізми реакції 12С(15N,14N)13C при енергії 81 МеВ

The new experimental data of the 12С(15N,14N)13С reaction cross-sections at the energy Еlab(15N) = 81 MeV were measured for the ground and excited states of 14N and 13C nuclei. The experimental data were analyzed within the coupled-reaction-channels method (CRC) using channels-coupling scheme with the 15N + 12С elastic scattering and one- as well as two-step transfers of nucleons and clusters performing CRC-calculations with the Woods - Saxon potentials (WS) for the entrance and exit reaction channels. The WS potential parameters for the 12С + 15N nuclear interaction were taken from the previously studied elastic and inelastic scattering of 15N ions by 12С nuclei at the energy 81 MeV, and the WS potential parameters for the 14N + 13С reaction exit channel were deduced from fitting of the 12С(15N,14N)13C reaction data. The spectroscopic factors (amplitudes) of transferred in the reaction nucleons and clusters, used in the CRC-calculations, were computed within translational invariant shell model of 1p-shell. It was found that transfers of neutrons (n) and deuterons (d) dominate in this reaction. Multi-step transfers of nucleons and clusters give small contributions to the reaction data.

Giant GAL gene clusters for the melibiose-galactose pathway in Torulaspora.

In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene cluster. We report here that some yeast strains in the genus Torulaspora have much larger GAL clusters that include genes for melibiase (MEL1), galactose permease (GAL2), glucose transporter (HGT1), phosphoglucomutase (PGM1) and the transcription factor GAL4, in addition to GAL1, GAL10, and GAL7. Together, these eight genes encode almost all the steps in the pathway for catabolism of extracellular melibiose (a disaccharide of galactose and glucose). We show that a progenitor 5‐gene cluster containing GAL 7‐1‐10‐4‐2 was likely present in the common ancestor of Torulaspora and Zygotorulaspora. It added PGM1 and MEL1 in the ancestor of most Torulaspora species. It underwent further expansion in the T. pretoriensis clade, involving the fusion of three progenitor clusters in tandem and the gain of HGT1. These giant GAL clusters are highly polymorphic in structure, and subject to horizontal transfers, pseudogenization and gene losses. We identify recent horizontal transfers of complete GAL clusters from T. franciscae into one strain of T. delbrueckii, and from a relative of T. maleeae into one strain of T. globosa. The variability and dynamic evolution of GAL clusters in Torulaspora indicates that there is strong natural selection on the GAL pathway in this genus.

Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron-sulfur cluster biosynthesis.

IscU is a central component of the ISC machinery and serves as a scaffold for de novo assembly of Fe-S clusters. The dedicated chaperone system composed of the Hsp70-chaperone HscA and the J-protein cochaperone HscB synergistically interacts with IscU and facilitates cluster transfer from IscU to recipient apo-proteins. Here, we report that the otherwise essential roles of HscA and HscB can be bypassed in vivo by a number of single amino acid substitutions in IscU. CD spectroscopic studies of the variant IscU proteins capable of this bypass activity revealed dynamic interconversion between two conformations: the denatured (D) and the structured (S) state in the absence and presence of Zn2+ , respectively, which was far more prominent than interconversion observed in wild-type IscU. Furthermore, we found that neither the S-shifted (more structured) variants of IscU nor the perpetually denatured variants could perform their in vivo role regardless of whether the chaperone system was present or not. The present study thus provides for the first time evidence that an in vivo D-state of IscU exists and implies that conformational interconversion between the S- and D-states of the scaffolding protein is a fundamental requirement for the assembly and transfer of the Fe-S cluster.

The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation.

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]2+ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]2+ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]2+ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]2+ clusters, via two-electron reductive coupling of two [2Fe-2S]2+ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.

4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]2+ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]2+ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]2+ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]2+ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]2+ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.