Preparation of a glycosynthase from the β-glycosidase of the Archaeon Pyrococcus horikoshii

The β-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phoβ-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phoβ-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.     

Physics and the canalization of morphogenesis: a grand challenge in organismal biology

Morphogenesis takes place against a background of organism-to-organism and environmental variation. Therefore, fundamental questions in the study of morphogenesis include: How are the mechanical processes of tissue movement and deformation affected by that variability, and in turn, how do the mechanic of the system modulate phenotypic variation? We highlight a few key factors, including environmental temperature, embryo size and environmental chemistry that might perturb the mechanics of morphogenesis in natural populations. Then we discuss several ways in which mechanics-including feedback from mechanical cues-might influence intra-specific variation in morphogenesis. To understand morphogenesis it will be necessary to consider whole-organism, environment and evolutionary scales because these larger scales present the challenges that developmental mechanisms have evolved to cope with. Studying the variation organisms express and the variation organisms experience will aid in deciphering the causes of birth defects.     

Infrared study of trifluoroacetic acid unpurified synthetic peptides in aqueous solution: trifluoroacetic acid removal and band assignment

Synthetic peptide or protein samples are mostly unpurified with trifluoroacetic acid (TFA) used during the synthesis procedure, which strongly interferes with structure determination by infrared (IR) spectroscopy. The aim of this work was to propose a simple strategy to remove TFA contribution from attenuated total reflection (ATR)–IR spectra of the hexahistidine peptide (His6) in aqueous solution to study the conformation of this synthetic peptide without previous purification. Such a strategy is based on the subtraction mode widely employed to remove water contribution, and it is tested with TFA unpurified histidine as a model system. The subtraction is based on eliminating the strong TFA bands at 1147 and 1200 cm⁻¹ by applying a scaling factor (as in buffer correction). The proposed modes represent excellent strategies that do not modify spectral features, and they provide reliable routines to obtain the synthetic peptide spectrum without TFA contribution. The conformational information from the corrected spectra at different pH values is deduced from semiempirical calculated IR spectra of different His6 conformers. The spectral features and the band positions of the corrected spectrum suggest that the peptide molecules mainly adopt an intermolecular β-sheet structure.     

Detailed characterization of the lipid A fraction from the nonpathogen Acinetobacter radioresistens strain S13

The genus Acinetobacter is composed of ubiquitous, generally nonpathogen environmental bacteria. Interest concerning these microorganisms has increased during the last 30 years, because some strains, belonging to the so-called A. baumannii-A. calcoaceticus complex, have been implicated in some severe pathological states in debilitated and hospitalized patients. The involvement of lipopolysaccharides (LPSs) as virulence factors in infections by Acinetobacter has been proven, and ongoing studies are aimed toward the complete serological characterization of the O-polysaccharides from LPSs isolated in clinical samples. Conversely, no characterization of the lipid A fraction from Acinetobacter strains has been performed. Here, the detailed structure of the lipid A fraction from A. radioresistens S13 is reported for the first time. A. radioresistens strains have never been isolated in cases of infectious disease. Nevertheless, it is known that the lipid A structure, with minor variations, is highly conserved across the genus; thus, structural details acquired from studies of this nonpathogen strain represent a useful basis for further studies of pathogen species.     

The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.     

Mitochondrial ND5 gene variation associated with encephalomyopathy and mitochondrial ATP consumption

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A-->G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (Deltapsim) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.     

Mitochondrial DNA-related Disorders

Mitochondrial diseases are a group of disorders due to a mitochondrial respiratory chain deficiency. They may depend on mitochondrial genome (mtDNA-related disorders) as well as on a nuclear genome defect (nDNA-related disorders). mtDNA-related disorders encompass an increasing number of clinical pictures associated with more than 250 different provisional or confirmed pathogenic changes in mtDNA. Although some clinical syndromes are nosologically defined, most of the cases present with polymorphous phenotypes ranging from pure myopathy to multi-system involvement. Complexity of mitochondrial genetics is in part responsible for the extreme clinical intra- and inter-familial heterogeneity of this group of diseases. In this review, we briefly report an updated classification and overview the main clinical pictures of this class of diseases.     

Mitochondrial Diseases: Therapeutic Approaches

Therapy of mitochondrial encephalomyopathies (defined restrictively as defects of the mitochondrial respiratory chain) is woefully inadequate, despite great progress in our understanding of the molecular bases of these disorders. In this review, we consider sequentially several different therapeutic approaches. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10. There is increasing interest in the administration of reactive oxygen species scavengers both in primary mitochondrial diseases and in neurodegenerative diseases directly or indirectly related to mitochondrial dysfunction. Aerobic exercise and physical therapy prevent or correct deconditioning and improve exercise tolerance in patients with mitochondrial myopathies due to mitochondrial DNA (mtDNA) mutations. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but interesting experimental approaches are being pursued and include, for example, decreasing the ratio of mutant to wild-type mitochondrial genomes (gene shifting), converting mutated mtDNA genes into normal nuclear DNA genes (allotopic expression), importing cognate genes from other species, or correcting mtDNA mutations with specific restriction endonucleases. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is becoming increasingly important for nuclear DNA-related disorders. Progress in each of these approaches provides some glimmer of hope for the future, although much work remains to be done.