Arnab Ghosh

Fe-S cluster biogenesis in isolated mammalian mitochondria: coordinated use of persulfide sulfur and iron, and requirements for GTP, NADH, and ATP

Background: Fe-S cluster assembly in mitochondria involves generation of an activated form of sulfur called persulfide. Results: A novel experimental system tracks new Fe-S cluster synthesis in isolated mammalian mitochondria. Conclusion: The use of persulfide sulfur and iron for Fe-S cluster biogenesis is tightly coordinated by processes requiring GTP, NADH, and ATP. Significance: These cofactor targets can now be defined. Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMPLICATIONS FOR ENZYME MATURATION*

Background: hsp90β binds to heme-free sGCβ1 subunit to enable heme insertion during maturation, but the mechanism is unclear. Results: Their interaction involves the hsp90β M domain and the Per-Arnt-Sim (PAS) domain of apo-sGCβ1. Conclusion: hsp90β may modulate the apo-sGCβ1 through its PAS domain, suggesting a means to aid heme insertion. Significance: This work expands our knowledge of how hsp90 regulates PAS-containing client proteins like sGC. Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGC) β1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90β and apo-sGCβ1. hsp90β and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCβ1 (sGCβ1(1–359)-H105F). Surprisingly, hsp90β and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-β1(1–359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90β M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCβ1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90β interaction could prevent apo-sGCβ1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.

Highly Active 2D Layered MoS 2 - rGO Hybrids for Energy Conversion and Storage Applications

The development of efficient materials for the generation and storage of renewable energy is now an urgent task for future energy demand. In this report, molybdenum disulphide hollow sphere (MoS2-HS) and its reduced graphene oxide hybrid (rGO/MoS2-S) have been synthesized and explored for energy generation and storage applications. The surface morphology, crystallinity and elemental composition of the as-synthesized materials have been thoroughly analysed. Inspired by the fascinating morphology of the MoS2-HS and rGO/MoS2-S materials, the electrochemical performance towards hydrogen evolution and supercapacitor has been demonstrated. The rGO/MoS2-S shows enhanced gravimetric capacitance values (318 ± 14 Fg−1) with higher specific energy/power outputs (44.1 ± 2.1 Whkg−1 and 159.16 ± 7.0 Wkg−1) and better cyclic performances (82 ± 0.95% even after 5000 cycles). Further, a prototype of the supercapacitor in a coin cell configuration has been fabricated and demonstrated towards powering a LED. The unique balance of exposed edge site and electrical conductivity of rGO/MoS2-S shows remarkably superior HER performances with lower onset over potential (0.16 ± 0.05 V), lower Tafel slope (75 ± 4 mVdec−1), higher exchange current density (0.072 ± 0.023 mAcm−2) and higher TOF (1.47 ± 0.085 s−1) values. The dual performance of the rGO/MoS2-S substantiates the promising application for hydrogen generation and supercapacitor application of interest.

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

ABSTRACT Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.

Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate reaction precisely recapitulated the 25S rRNA cleavage pattern observed in cells, indicating that redox activity of the ribosome-bound iron is responsible for the strand breaks in the rRNA. In summary, our findings provide evidence that oxidative stress–associated rRNA cleavages can occur through rRNA strand scission by redox-active, ribosome-bound iron that potentially promotes Fenton reaction–induced hydroxyl radical production, implicating intracellular iron as a key determinant of the effects of oxidative stress on ribosomes. We propose that iron binding to specific ribosome elements primes rRNA for cleavages that may play a role in redox-sensitive tuning of the ribosome function in stressed cells.

In situ synchrotron X-ray diffraction study of coherently embedded silver nanostructure growth in silicon

We report on the in situ growth of coherently embedded Ag nanostructures using real time temperature dependent synchrotron X-ray diffraction (XRD) measurements. ≈17 nm thick GeOx film was grown on native oxide covered silicon substrates (GeOx/SiOx/Si) using a physical vapor deposition (PVD) method, which were used as the substrates for Ag nanostructures growth. For growing Ag nanostructures, two different sources of silver were used. In one system, ≈2 nm silver thin film was grown on the GeOx/SiOx/Si substrates using a PVD method, while in another system, silver wires were kept on the specimen hot stage (chemical vapor deposition) along with the above substrates. All the in situ growth and real time XRD were done under atmospheric conditions. The lattice constant of the Ag nanostructures obtained from the ex situ growth specimens was used to compare with the real time high temperature XRD measurements. As the temperature is raised from room temperature to 850 °C while performing in situ growth, the evolutions of various diffraction peaks such as (111), (200) and (220), reflecting from the growth facets of Ag nanostructures, were monitored. By measuring the deviation of the Ag lattice parameter due to the shift in the diffraction peak positions as a function of temperature, the thermal expansion coefficients for the Ag nanostructures in a matrix have been determined. In one case, the thermal expansion coefficient was found to decrease from 1.9 × 10−5/°C to 1.82 × 10−5/°C with the increase of annealing temperature from 750 °C to 850 °C.