Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Biochemical composition of two red seaweed species grown on the Brazilian coast

BACKGROUND: Algae species have been used as an important source of food because they are highly nutritive considering their vitamin, protein, mineral, fiber, essential fatty acid and carbohydrate contents. However, a large number of seaweeds have been poorly studied, especially Brazilian species. Two red macroalgae species from the Brazilian coast (Plocamium brasiliense and Ochtodes secundiramea) were assessed with respect to their total lipid, fatty acid, total nitrogen, protein, amino acid and total carbohydrate contents. RESULTS: The total lipid contents (dry weight) were 36.3 and 35.4 g kg(-1); fatty acid contents were 9.3 and 12.1 g kg(-1); total nitrogen contents were 37.4 and 24.9 g kg(-1); protein contents were 157.2 and 101.0 g kg(-1); amino acid contents were 127.5 and 91.4 g kg(-1); and total carbohydrate contents were 520.3 and 450.7 g kg(-1) for P. brasiliense and O. secundiramea, respectively. CONCLUSION: Considering these compositions, both algae species were determined to have sources of protein, essential amino acids and carbohydrates similar to the edible seaweeds Laminaria japonica and Palmaria palmata. (C) 2011 Society of Chemical Industry

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.

Sodium Tungstate on Some Biochemical Parameters of the Parotid Salivary Gland of Streptozotocin-Induced Diabetic Rats: A Short-Term Study

Several studies have shown the antidiabetic properties of sodium tungstate. In this study, we evaluated some biochemical parameters of the parotid salivary gland of streptozotocin-induced diabetic rats treated with sodium tungstate solution (2 mg/ml). The studied groups were: untreated control (UC), treated control (TC), untreated diabetic (UD), and treated diabetic (TD). After 2 and 6 weeks of treatment, parotid gland was removed and total protein and sialic acid (free and total) concentration and amylase and peroxidase activities were determined. Data were compared by variance analysis and Tukey test (p < 0.05). The sodium tungstate treatment modestly decreased the glycemia of streptozotocin-induced diabetic rats. At week 2 of the study, parotid gland of diabetic rats presented a reduction of total protein concentration (55%) and an increase of amylase (120%) and peroxidase (160%) activities, free (150%) and total (170%) sialic acid concentration. No alteration in the evaluated parameters at week 6 of the study was observed. Sodium tungstate presented no significant effect in parotid gland. Our results suggest that diabetes causes initial modification in biochemical composition of parotid. However, this gland showed a recovery capacity after 6 week of the experimental time. Sodium tungstate has no effect in peripheral tissues, such as salivary glands.

Structure, Dynamics, and RNA Interaction Analysis of the Human SBDS Protein

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS. (C) 2010 Elsevier Ltd. All rights reserved.

Sdo1p, the yeast orthologue of Shwachman-Bodian-Diamond syndrome protein, binds RNA and interacts with nuclear rRNA-processing factors

The Shwachman-Bodian-Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre-60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co-immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright (C) 2009 John Wiley & Sons, Ltd.

Oroidin Inhibits the Activity of the Multidrug Resistance Target Pdr5p from Yeast Plasma Membranes

Oroidin was isolated from the marine sponge Agelas sventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.

Human regulatory protein Ki-1/57 has characteristics of an intrinsically unstructured protein

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1; in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.