Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monte Carlo and modified Tanford-Kirkwood results for macromolecular electrostatics calculations

The understanding of electrostatic interactions is an essential aspect of the complex correlation between structure and function of biological macromolecules. It is also important in protein engineering and design. Theoretical studies of such interactions are predominantly done within the framework of Debye-Huckel theory. A classical example is the Tanford-Kirkwood (TK) model. Besides other limitations, this model assumes an infinitesimally small macromolecule concentration. By comparison to Monte Carlo (MC) simulations, it is shown that TK predictions for the shifts in ion binding constants upon addition of salt become less reliable even at moderately macromolecular concentrations. A simple modification based on colloidal literature is suggested to the TK scheme. The modified TK models suggested here satisfactorily predict MC and experimental shifts in the calcium binding constant as a function of protein concentration for the calbindin D-9k mutant and calmodulin.

Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues

Antimicrobial peptides (AMPs) are effector molecules of innate immune systems found in different groups of organisms, including microorganisms, plants, insects, amphibians and humans. These peptides exhibit several structural motifs but the most abundant AMPs assume an amphipathic alpha-helical structure. The alpha-helix forming antimicrobial peptides are excellent candidates for protein engineering leading to an optimization of their biological activity and target specificity. Nowadays several approaches are available and this review deals with the use of combinatorial synthesis and directed evolution in order to provide a high-throughput source of antimicrobial peptides analogues with enhanced lytic activity and specificity.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; λmax = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; λmax = 623 nm) red light-emitting luciferases. The emission spectrum (λmax = 571 nm) and KM for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 226-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (λmax = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

Caracterização estrutural e biofísica de duas proteínas relacionadas: β-1,3-1,4-glicanase de Bacillus subtilis 168 e α-L-arabinofuranosidase termoestável de Thermotoga petrophila RKU-1

Pós-graduação em Biofísica Molecular - IBILCE

Estabilização de enzimas: uma abordagem

Enzyme stabilization is one critic point in basic and applied enzymology. The increasing interest in applying enzymes in industrial processes has fostered the search for biocatalysts with new properties or extreme stability. Enzyme stabilization can be achieved by different methods: isolating enzyme variants from organisms living in appropriate extreme environments (extremozymes), by protein engineering, chemical modification, use of additives, immobilization. This brief review aims to give a better understanding of those methods employed for enzyme stabilization.

Production of active recombinant eIF5A: reconstitution in E. coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

PDGF-B gene therapy accelerates bone engineering and oral implant osseointegration

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 x 10(8) or 5.5 x 10(9) pfu ml (1)), Ad encoding luciferase (Ad-Luc; 5.5 x 10(9) pfu ml (1); control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml (1)). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, microcomputed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo. Gene Therapy (2010) 17, 95-104; doi: 10.1038/gt.2009.117; published online 10 September 2009

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bacterial cellulose-collagen nanocomposite for bone tissue engineering

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of atrazine on female Wistar rats: Morphological alterations in ovarian follicles and immunocytochemical labeling of 90 kDa heat shock protein

Fertility in female mammals may be affected by a variety of endocrine disrupters present in the environment. Herbicide atrazine is an example of endocrine disrupter employed in agriculture, which disrupts estrous cyclicity in rats. Aiming to characterize morphologically the effect of low and sublethal doses of atrazine on the ovaries of Wistar rats, in an effort to determine the possible intrafollicular target site through which this herbicide acts adult females were submitted to both subacute and subchronic treatments. Additionally, immunocytochemical labeling of 90 kDa heat shock protein (HSP90) was performed in order to evaluate the role played by this protein in the ovary, under stressed conditions induced by herbicide exposure. The results indicated that atrazine induced impaired folliculogenesis, increased follicular atresia and HSP90 depletion in female rats submitted to subacute treatment, while the subchronic treatment with low dose of atrazine could compromise the reproductive capacity reflected by the presence of multioocytic follicle and stress-inducible HSP90. © 2007 Elsevier Ltd. All rights reserved.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Influence of salts on the coexistence curve and protein partitioning in nonionic aqueous two-phase micellar systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)