FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants

There is great interest in increasing proteins’ stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt’s reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔT _m = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.     

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

BackgroundsSpontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.MethodsPCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.ResultsThe 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.General significanceIdentification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.     

Apolipoprotein-J prevention of fetal cardiac myoblast apoptosis induced by ethanol

Over-consumption of ethanol (EtOH) represents a major health problem. This study was to test the cytotoxicity of EtOH in cardiac stem cells or myoblasts, and the potential protective effect of apolipoprotein-J (ApoJ), a stress-responding, chaperone-like protein in high-density lipoprotein, on EtOH-injured cardiac myoblasts. In culture, EtOH-exposed canine fetal myoblasts underwent apoptosis in a concentration- and time-dependent manner. Expression ApoJ by cDNA transfection markedly reduced EtOH-induced apoptosis in the cells. ApoJ expression also restored partially the mitochondrial membrane potential and prevented the release of cytochrome-c from mitochondria into cytoplasma. Thus, ApoJ serves as a cytoprotective protein that protects cardiac stem cells against EtOH cytotoxicity.     

Role of stem cells in cancer therapy and cancer stem cells: a review

For over 30 years, stem cells have been used in the replenishment of blood and immune systems damaged by the cancer cells or during treatment of cancer by chemotherapy or radiotherapy. Apart from their use in the immuno-reconstitution, the stem cells have been reported to contribute in the tissue regeneration and as delivery vehicles in the cancer treatments. The recent concept of 'cancer stem cells' has directed scientific communities towards a different wide new area of research field and possible potential future treatment modalities for the cancer. Aim of this review is primarily focus on the recent developments in the use of the stem cells in the cancer treatments, then to discuss the cancer stem cells, now considered as backbone in the development of the cancer; and their role in carcinogenesis and their implications in the development of possible new cancer treatment options in future.     

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats

Molecular Biology Reports - On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose...     

The impact of read length on quantification of differentially expressed genes and splice junction detection

BackgroundThe initial next-generation sequencing technologies produced reads of 25 or 36 bp, and only from a single-end of the library sequence. Currently, it is possible to reliably produce 300 bp paired-end sequences for RNA expression analysis. While read lengths have consistently increased, people have assumed that longer reads are more informative and that paired-end reads produce better results than single-end reads. We used paired-end 101 bp reads and trimmed them to simulate different read lengths, and also separated the pairs to produce single-end reads. For each read length and paired status, we evaluated differential expression levels between two standard samples and compared the results to those obtained by qPCR.ResultsWe found that, with the exception of 25 bp reads, there is little difference for the detection of differential expression regardless of the read length. Once single-end reads are at a length of 50 bp, the results do not change substantially for any level up to, and including, 100 bp paired-end. However, splice junction detection significantly improves as the read length increases with 100 bp paired-end showing the best performance. We performed the same analysis on two ENCODE samples and found consistent results confirming that our conclusions have broad application.ConclusionsA researcher could save substantial resources by using 50 bp single-end reads for differential expression analysis instead of using longer reads. However, splicing detection is unquestionably improved by paired-end and longer reads. Therefore, an appropriate read length should be used based on the final goal of the study.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0697-y) contains supplementary material, which is available to authorized users.     

Purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures

The present study is focused on purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures--hormidium sp. A27 DM, Lyngbya sp. A09 DM and Halomicronema sp. A32 DM. 'Phycoerythrin' was successfully purified and characterized. On SDS-PAGE, the PE purified from all three young cultures showed four bands--corresponding to α and β subunits of each of PE-I and PE-II. However, phycoerythrin purified after prolonged growth of Phormidium sp. A27 DM and Halomicronema sp. A32DM showed only one band corresponding to 14 kDa whereas Lyngbya sp. A09 DM continued to produce uncleaved phycoerythrin. The absorption spectra of purified PEs from all the three young and old cultures showed variations however the fluorescence studies of the purified PEs in all cases gave the emission spectra at around 580 nm. The described work is of great importance to understand the role of phycoerythrin in adapting cyanobacteria to stress conditions.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Toward locating the source of serotonergic axons in the tail nerve of <Emphasis Type="Italic">Aplysia</Emphasis>

Stimulation of the tail nerve (pedal nerve 9, p9) of the mollusk, Aplysia californica, causes release of serotonin (5-HT), which mediates sensitization of withdrawal responses. There are about 35 serotonin-immunoreactive (5-HT-ir) axons in p9, yet the cell bodies of these axons have not been located. Backfills of p9 were combined with 5-HT immunohistochemistry to locate the cell bodies of 5-HT-ir neurons with axons in p9. About 100 neurons had axons in p9. Only about ten neurons, however, were both backfilled and 5-HT-ir. These double-labeled neurons were all located in the pedal ganglion associated with p9, which had a total of approximately 42 5-HT-ir somata. The discrepancy between the number of 5-HT-ir axons and double-labeled cell bodies is not likely due to neurons having multiple axons in the nerve; intracellular fills suggest that these neurons do not branch before entering p9. Additionally, no evidence was found for peripheral 5-HT-ir cell bodies that project axons centrally through p9. Thus, approximately 70% of the neurons that give rise to the 5-HT-ir axons in tail nerve are unaccounted for, but likely to reside in the pedal ganglion.