Fine-tuning of the Hsc70-based Human Protein Disaggregase Machinery by the Distinctive C-terminal Extension of Apg2

Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain, with propensity to adopt α-helical structure, interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system.     

Settling-site selection and survival of two scale insects,Ceroplastes rubens andC. ceriferus,on citrus trees

We studied settling-site selection and the resulting survival of two sessile scale insects, Ceroplastes rubens and C. ceriferus, in the citrus tree, Citrus unshiu, in central Japan. C. rubens preferred 0-year-old twigs most as a settling-site; the density of nymphs settling on 0-year-old twigs was significantly higher than those on ≥1-year-old twigs, and few nymphs settled on ≥3-year-old twigs. The mean survival rates from settling until reproduction in the next year were significantly higher on more preferred twigs than on less preferred ones. In C. ceriferus, nymphs significantly preferred 1- and 2-year-old twigs to 0- and ≥3-year-old ones, and the mean survival rates on the more preferred 1- and 2-year-old twigs were significantly higher than those on less preferred ≥3-year-old twigs. However, the survival rate on less preferred 0-year-old twigs was slightly higher than those on 1- and 2-year-old ones. Thus, in both species of scale, it was the preferred twigs which were more profitable sites for survival after settling, except for less preferred 0-year-old twigs for C. ceriferus. In both scale species, most mortality was due to growth cessation, which is believed to be related to the twig quality as a food source. Predators and parasitoids were minor mortality factors. Both species showed constant survival rates until the density of settled nymphs exceeded double the “upper-limit” density, whereupon they decreased drastically. Nymphs of C. rubens settling on twigs of high scale density showed a spacing-out distribution, those of C. ceriferus did not. In C. rubens, an increase in preference for originally less profitable twigs at the later stage of the settling season was observed, but not in C. ceriferus. Accordingly, individuals of C. rubens showed a stronger tendency to avoid conspecifics than did C. ceriferus. Although nymphs of the two scales clearly preferred more profitable sites, their settling-site selection did not agree with the predictions from the ideal free distribution theory (Fretwell and Lucas, 1970). The discrepancies were (1) frequent settling on less profitable sites at the early stage of the settling season, (2) insufficient utilization of the most profitable twigs, and (3) virtually 100% mortality on overcrowded twigs under conditions where unoccupied profitable twigs still remained. These discrepancies are thought due to the limited dispersal time of nymphs.     

Structural variability of C3larvin toxin. Intrinsic dynamics of the α/β fold of the C3-like group of mono-ADP-ribosyltransferase toxins

C3larvin toxin is a new member of the C3 class of the mono-ADP-ribosyltransferase toxin family. The C3 toxins are known to covalently modify small G-proteins, e.g. RhoA, impairing their function, and serving as virulence factors for an offending pathogen. A full-length X-ray structure of C3larvin (2.3 Å) revealed that the characteristic mixed α/β fold consists of a central β-core flanked by two helical regions. Topologically, the protein can be separated into N and C lobes, each formed by a β-sheet and an α-motif, and connected by exposed loops involved in the recognition, binding, and catalysis of the toxin/enzyme, i.e. the ADP-ribosylation turn–turn and phosphate–nicotinamide PN loops. Herein, we provide two new C3larvin X-ray structures and present a systematic study of the toxin dynamics by first analyzing the experimental variability of the X-ray data-set followed by contrasting those results with theoretical predictions based on Elastic Network Models (GNM and ANM). We identify residues that participate in the stability of the N-lobe, putative hinges at loop residues, and energy-favored deformation vectors compatible with conformational changes of the key loops and 3D-subdomains (N/C-lobes), among the X-ray structures. We analyze a larger ensemble of known C3bot1 conformations and conclude that the characteristic ‘crab-claw’ movement may be driven by the main intrinsic modes of motion. Finally, via computational simulations, we identify harmonic and anharmonic fluctuations that might define the C3larvin ‘native state.’ Implications for docking protocols are derived.     

Analysis of potassium iodate reduction in tissue homogenates using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

Potassium iodate (KIO₃) and potassium iodide (KI) are the major salt iodization agents used worldwide. Unlike iodide (I⁻), iodate (IO₃⁻) should be reduced to I⁻ before it can be effectively used by the thyroid. In this study, we developed a new method for analyzing IO₃⁻ and I⁻ in tissue homogenates using high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC–ICP-MS). We further applied the method to demonstrate the KIO₃ reduction process by tissues in vitro. The effects of KIO₃ on the total antioxidative activity (TAA) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were also investigated here. Finally, we found that IO₃⁻ can be reduced to I⁻ by tissue homogenates and IO₃⁻ irreversibly decreases the antioxidant capability of tissues. Our studies suggest that KIO₃ might have a big effect on the redox balance of tissue and would further result in oxidative stress of organisms.     

The role of the medium in long-range electron transfer

 The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism. Received: 2 January 1997 / Accepted: 6 February 1997     

Estimating the human appropriation of land in Brazil by means of an Input–Output Economic Model and Ecological Footprint analysis

As we confront the current environmental crisis, determining the biophysical base (e.g., materials, energy, land, and water) of nations has become paramount. With advanced economies benefiting from the import of resource-intensive primary goods originating from poorer parts of the world, especially emerging nations, these are dilapidating their natural capital. Brazil is one of such emerging economies, whose mining and farming activities, propping up its export-led economic growth, exert great pressure on the environment. In particular, farming has been shown to have one of the world's greatest environmental impacts, especially as a consequence of land use associated with cattle ranching. Since a nation-wide evaluation of land-use types across the whole sectorial spectrum of the country's economy is still lacking, we used the most recently available Input–Output Economic Model for Brazil and the Ecological Footprint method to identify those economic sectors with the greatest potential for appropriating portions of the natural world.Our results show that: (i) the biggest chunk of Brazil's Ecological Footprint is due to its Carbon Footprint and, in particular, emissions from cattle; (ii) only a few economic sectors exhibit high Ecological Footprint values, chiefly those belonging to livestock farming and energy production based on fossil fuels; (iii) excluding the soybeans and slaughter sectors, export-oriented sectors have below-average Ecological Footprint values; and (iv) the percentage of Brazil's Ecological Footprint due to household consumption (excluding imports) is three times bigger than that attributable to exports, with sectors belonging to livestock farming contributing the most to such disparity.These results underscore that the environmental impact of the Brazilian economy can be drastically reduced by tackling the emission-intensive production processes of a few sectors only and disincentivizing the domestic consumption of a narrow range of products, especially with respect to the livestock segment.     

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

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