Human myocardial Na,K-ATPase concentration in heart failure

The Na,K-ATPase is of major importance for active ion transport across the sarcolemma and thus for electrical as well as contractile function of the myocardium. Furthermore, it is receptor for digitalis glycosides. In human studies of the regulatory aspects of myocardial Na,K-ATPase concentration a major problem has been to obtain tissue samples. Methodological accomplishments in quantification of myocardial Na,K-ATPase using vanadate facilitated ³H-ouabain binding to intact samples have, however, made it possible to obtain reliable measurements on human myocardial necropsies obtained at autopsy as well as on biopsies of a wet weight of only 1–2 mg obtained during heart catheterisation. However, access to the ultimately, normal, vital myocardial tissue has come from the heart transplantation programs, through which myocardial samples from cardiovascular healthy organ donors have become available. In the present paper we evaluate the various values reported for normal human myocardial Na,K-ATPase concentration, its regulation in heart disease and the association with digitalization. Normal myocardial Na,K-ATPase concentration level is found to be 700 pmol/g wet weight. No major variations were found between or within the walls of the heart ventricles. During the first few years of life a marked decrease in myocardial Na,K-ATPase concentration is followed by a stable level obtained in early adulthood and normally maintained throughout life. In patients with enlarged cardiac x-ray silhouette a significant positive, linear correlation between left ventricular ejection fraction (EF) and Na,K-ATPase concentration was established. A maximum reduction in Na,K-ATPase concentration of 89% was obtained when EF was reduced to 20%. Generally, heart failure associated with heart dilatation, myocardial hypertrophy as well as ischaemic heart disease is associated with reductions in myocardial Na,K-ATPase concentration of around 25%. During digoxin treatment of heart failure patients a further reduction in functional myocardial Na,K-ATPase concentration of 15% has been found. Thus, the total reduction in functional myocardial Na,K-ATPase concentration in digitalised heart failure patients may well be of the magnitude 40%. In conclusion, it has become possible to quantify human myocardial Na,K-ATPase in health and disease. Revealed reductions are in heart failure of importance for contractile function, generation of arrhythmia and for digoxin treatment.     

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.