Paulo R. Dores-Silva

Processo de estabilização de resíduos orgânicos: vermicompostagem versus compostagem

Two processes are used to stabilize organic wastes: composting and vermicomposting.There are no studies in the literature showing which process is most effective over the short term. In this study, 3 organic wastes were composted and vermicomposted for 90 days, and the parameters pH, effective cation exchange capacity, total organic carbon, total Kjeldahl nitrogen, Ptotal, E4/E6 ratio, hydrophobicity and aromaticity indexes were determined. In all experiments, vermicomposted materials showed higher stability, proving a superior tool for stabilization of these organic wastes.

Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

Structural and stability studies of the human mtHsp70-escort protein 1: an essential mortalin co-chaperone.

Mitochondrial Hsp70 is involved in both protein import and folding process, among other essential functions. In mammalian cells, due to its role in the malignant process, it receives the name of mortalin. Despite its importance in protein and mitochondrial homeostasis, mortalin tends to self-aggregate in vitro and in vivo, the later leads to mitochondrial biogenesis failure. Recently, a zinc-finger protein, named Hsp70-escort protein 1 (Hep1, also called Zim17/TIM15/DNLZ), was described as an essential human mitochondrial mortalin co-chaperone which avoids its self-aggregation. Here, we report structural studies of the human Hep1 (hHep1). The results indicate that hHep1 shares some structural similarities with the yeast ortholog despite the low identity and functional differences. We also observed that hHep1 oligomerizes in a concentration-dependent fashion and that the zinc ion, which is essential for hHep1 in vivo function, has an important protein-structure stabilizing effect.

A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering

Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.

Acompanhamento químico da vermicompostagem de lodo de esgoto doméstico

the final product (vermicompost) in agricultural soils. The monitored chemical variables during the 90 days of vermicomposting were: humidity rate, organic matter content, nitrogen and phosphorus content, pathogenic organisms concentration, total organic carbon, acidity, CEC, C/N ratio, CEC/TOC ratio, and humic and fulvic acids content. The change in these variables during the vermicomposting process showed that this technique is effective for use in the maturation of the residue.

Chemical Differentiation of Domestic Sewage Sludge and Cattle Manure Stabilized by Microbioreators: Study by Pyrolysis Coupled to Gas Chromatography Coupled to Mass Spectroscopy

The aim of the present study was to evaluate the chemical alterations in substrates of vermicomposts from domestic sewage sludge and from cattle manure, besides its quality as an organic fertilizer. Elemental and infrared analysis and UV-Vis spectroscopy were used for their characterizations. Chemical characteristics determined in the vermicomposts indicate that they can be used as organic fertilizers, mainly with regard to organic matter content, pH, C/N ratio, and nitrogen and phosphorus levels. The main constituents of the humic substances extracted from the vermicomposts were obtained by pyrolysis coupled to gas chromatography-mass spectrometry (GC/MS), showing that these compounds are quite similar, despite being produced by domestic sewage sludges or by cattle manure.

Limited proteolysis of myoglobin opens channel in ferrochelatase-globin complex for iron to zinc transmetallation

Recombinant ferrochelatase (BsFECH) from Bacillus subtilis expressed in Escherichia coli BL21(DE3) was found by UV-visible spectroscopy to bind the model substrate tetraphenylporphyrin-sulfonate, TPPS, with Ka=3.8 10(5)mol/L in aqueous phosphate buffer pH 5.7 at 30°C, and to interact with metmyoglobin with Ka=1.07±0.13 10(5)mol/L at 30°C. The iron/zinc exchange in myoglobin occurring during maturation of Parma hams seems to depend on such substrate binding to BsFECH and was facilitated by limited pepsin proteolysis of myoglobin to open a reaction channel for metal exchange still with BsFECH associated to globin. BsFECH increased rate of zinc insertion in TPPS significantly and showed saturation kinetics with an apparent binding constant of Zn(II) to the [enzyme-TPPS] complex of 1.3 10(4)mol/L and a first-order rate constant of 6.6 10(-1)s(-1) for dissociation of the tertiary complex, a similar pattern was found for zinc/iron transmetallation in myoglobin.

Structural and functional studies of Hsp70-escort protein-Hep1-of Leishmania braziliensis

Hep1 is a mitochondrial Hsp70 (mtHsp70) co-chaperone that presents a zinc finger domain essential for its function. This co-chaperone acts to maintain mtHsp70 in its soluble and functional state. In this work, we have demonstrated that Leishmania braziliensis mtHsp70 (LbmtHsp70) is also dependent on the assistance of Hep1. To understand the L. braziliensis Hep1 (LbHep1) structure-function relationship, we produced LbHep1 and two truncated mutants corresponding to the C-terminal zinc finger domain and the N-terminal region. We observed that LbHep1 is composed of an unfolded N-terminal region and a β-sheet-folded C-terminal domain, which holds the zinc-binding motif. Both LbHep1 and the zinc finger domain construction maintained LbmtHsp70 solubility in co-expression systems after cell lysis. In solution, LbHep1 behaved as a highly elongated monomer, probably due to the unfolded N-terminal region. Furthermore, we also observed that the zinc ion interacted with LbHep1 with high affinity and was critical for LbHep1 structure and stability because its removal from LbHep1 solutions altered the protein structure and stability. In vitro, LbHep1 protected, in sub-stoichiometric fashion, LbmtHsp70 from thermally induced aggregation but did not present intrinsic chaperone activity on model client proteins. Therefore, LbHep1 is a specific chaperone for LbmtHsp70.

Insights on the structural dynamics of Leishmania braziliensis Hsp90 molecular chaperone by small angle X-ray scattering

Heat shock protein of 90kDa (Hsp90) is an essential molecular chaperone involved in a plethora of cellular activities which modulate protein homeostasis. During the Hsp90 mechanochemical cycle, it undergoes large conformational changes, oscillating between open and closed states. Although structural and conformational equilibria of prokaryotic and some eukaryotic Hsp90s are known, some protozoa Hsp90 structures and dynamics are poorly understood. In this study, we report the solution structure and conformational dynamics of Leishmania braziliensis Hsp90 (LbHsp90) investigated by small angle X-ray scattering (SAXS). The results indicate that LbHsp90 coexists in open and closed conformations in solution and that the linkers between domains are not randomly distributed. These findings noted interesting features of the LbHsp90 system, opening doors for further conformational studies of other protozoa chaperones.

Structural and functional studies of the Leishmania braziliensis SGT co-chaperone indicate that it shares structural features with HIP and can interact with both Hsp90 and Hsp70 with similar affinities

Molecular chaperones and co-chaperones play an essential role in the life cycles of protozoa belonging to the genus Leishmania. The small glutamine-rich TPR-containing protein (SGT) is a co-chaperone that can be divided into three domains: N-terminal, tetratricopeptide (TPR) and C-terminal. The TPR domain is responsible for interactions with both Hsp70 and Hsp90; however, the mechanism of interaction and the functionality of SGT are unclear. In this context, we present the structural and functional characterization of Leishmania braziliensis SGT (LbSGT), aiming to elucidate how this co-chaperone interacts with the Hsp90/Hsp70 chaperone machinery. Structurally, the recombinant LbSGT behaves as an α-helical, multidomain and elongated dimer in solution. Despite their low amino acid sequence identity and similarity, LbSGT shares structural properties and domain organization with the Hsp70-interacting protein (HIP) co-chaperone. Functionally, LbSGT is a cognate protein in L. braziliensis promastigote cells and interacts indiscriminately, with similar affinities, with both Hsp90 and Hsp70 chaperones, capable of working as an adaptor protein. Sequence analysis indicates that LbSGT interacts via a dicarboxylate clamp, the same mechanism used by the Hsp90-Hsp70-organizing protein (HOP) co-chaperone. These results suggest that SGT can develop the same function as HOP but using the HIP structural scaffold.