Evidence for a synergistic effect of post‐translational modifications and genomic composition of eEF‐1α on the adaptation of Phytophthora infestans

Genetic variation plays a fundamental role in pathogen''s adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post‐translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor‐1α (eEF‐lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF‐lα protein, possibly attributable to purifying selection and a lack of intra‐genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF‐1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF‐1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre‐ and post‐translational phase in eEF‐1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.     

A general synthetic method toward uridylylated picornavirus VPg proteins

Small proteins called viral protein genome‐linked (VPg), attached to the 5′‐end of the viral RNA genome are found as common structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre‐uridylylated tyrosine building block in an on‐line solid phase‐based approach. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Deep and quantitative top-down proteomics in clinical and translational research

It has long been understood that it is proteins, expressed and post-translationally modified, that are the primary regulators of both the fate and the function of cells. The ability to measure differences in the expression of the constellation of unique protein forms (proteoforms) with complete molecular specificity has the potential to sharply improve the return on investment for mass spectrometry-based proteomics in translational research and clinical diagnostics.     

ABCG2/BCRP Dysfunction as a Major Cause of Gout

Recent genome-wide association studies showed that serum uric acid (SUA) levels relate to ABCG2/BCRP gene, which locates in a gout-susceptibility locus revealed by a genome-wide linkage study. Together with the ABCG2 characteristics, we hypothesized that ABCG2 transports urate and its dysfunction causes hyperuricemia and gout. Transport assays showed ATP-dependent transport of urate via ABCG2. Kinetic analysis revealed that ABCG2 mediates high-capacity transport of urate (Km: 8.24 ± 1.44 mM) even under high-urate conditions. Mutation analysis of ABCG2 in 90 Japanese hyperuricemia patients detected six nonsynonymous mutations, including five dysfunctional variants. Two relatively frequent dysfunctional variants, Q126X and Q141K, were then examined. Quantitative trait locus analysis of 739 Japanese individuals showed that Q141K increased SUA as the number of minor alleles of Q141K increased (p = 6.60 × 10^?5). Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. Becuase Q126X and Q141K are assigned to nonfunctional and half-functional haplotypes, respectively, their genotype combinations are divided into four functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those with dysfunctional ABCG2 increased the gout risk, especially those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10^?21). These genotypes were found in 10.1% of gout patients, but in only 0.9% of control. Our function-based clinicogenetic (FBCG) analysis showed that combinations of the two dysfunctional variants are major causes of gout, thereby providing a new approach for prevention and treatment of the gout high-risk population.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.     

[PSI+] turns 50

abstract

The year 2015 sees the fiftieth anniversary of the publication of a research paper that underpins much of our understanding of fungal prion biology, namely “ψ, a cytoplasmic suppressor of super-suppressor in yeast” by Brian Cox. Here we show how our understanding of the molecular nature of the [PSI⁺] determinant evolved from an ‘occult’ determinant to a transmissible amyloid form of a translation termination factor. We also consider the impact studies on [PSI] have had – and continue to have - on prion research. To demonstrate this, leading investigators in the yeast prion field who have made extensive use of the [PSI⁺] trait in their research, provide their own commentaries on the discovery and significance of [PSI].     

Automated analysis of courtship suppression learning and memory in Drosophila melanogaster

Study of the fruit fly, Drosophila melanogaster, has yielded important insights into the underlying molecular mechanisms of learning and memory. Courtship conditioning is a well-established behavioral assay used to study Drosophila learning and memory. Here, we describe the development of software to analyze courtship suppression assay data that correctly identifies normal or abnormal learning and memory traits of individual flies. Development of this automated analysis software will significantly enhance our ability to use this assay in large-scale genetic screens and disease modeling. The software increases the consistency, objectivity, and types of data generated.