Contrast-FEL—A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.