The analysis of multiple O-phosphoseryl-containing peptides by fast atom bombardment mass spectrometry

Summary Positive and negative ion FAB mass spectrometry were found to be useful for the structural analysis of phosphorylated peptides containing multiple O-phosphoseryl residues. The positive ion FAB mass spectra obtained for Ac-Ser(P)-Ser(P)-NHMe and Ac-Ser(P)-Ser(P)-Ser(P)-NHMe showed that -eliminative loss of H₃PO₄ from the Ser(P)-residue was a major event in the fragmentation of the two phosphopeptides and that successive losses of H₃PO₄ from the [M+H]⁺ ion occurred when the Ser(P)-cluster was located at the N-terminus. In contrast, the FAB mass spectrum of Ac-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe showed only a single loss of H₃PO₄ from the [M+H]⁺ ion, with further losses of H₃PO₄ from internal Ser(P)-residues only occurring when fragmentation of the parent phosphopeptide generated daughter fragments that contained (part of) an N-terminal Ser(P)-residue. Negative ion FAB mass spectrometry also proved useful for the structural analysis of the three Ser(P)-peptides and showed high-intensity [M-H]^- ions along with minor [M-H-80]^- fragment ions.Abbreviations Ac acetyl - Ala dehydroalanyl - FAB-MS fast atom bombardment mass spectrometry - LSIMS liquid secondary ion mass spectrometry - NHMe N-methylamide - Ser(P) O-phosphoseryl - Thr(P) O-phosphothreonyl     

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 ¹⁵N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

Differential effects of serine side chain interactions in amyloid formation by islet amyloid polypeptide

Islet amyloid polypeptide (IAPP), a 37 residue polypeptide, is the main protein component of islet amyloid deposits produced in the pancreas in Type 2 diabetes. Human IAPP contains five serine residues at positions 19, 20, 28, 29, and 34. Models of the IAPP amyloid fibril indicate a structure composed of two closely aligned columns of IAPP monomers with each monomer contributing to two intermolecular β‐strands. Ser 19 and Ser 20 are in the partially ordered β‐turn region, which links the two strands, whereas Ser 28, Ser 29, and Ser 34 are in the core region of the amyloid fibril. Ser 29 is involved in contacts between the two columns of monomers and is the part of the steric zipper interface. We undertook a study of individual serine substitutions with the hydrophobic isostere 2‐aminobutyric acid (2‐Abu) to examine the site‐specific role of serine side chains in IAPP amyloid formation. All five variants formed amyloid. The Ser 19 to 2‐Abu mutant accelerates amyloid formation by a factor of 3 to 4, while the Ser 29 to 2‐Abu mutation modestly slows the rate of amyloid formation. 2‐Abu replacements at the other sites had even smaller effects. The data demonstrate that the cross‐column interactions made by residue 29 are not essential for amyloid formation and also show that cross‐strand networks of hydrogen‐bonded Ser side chains, so called Ser‐ladders, are not required for IAPP amyloid formation. The effect of the Ser 19 to 2‐Abu mutant suggests that residues in this region are important for amyloid formation by IAPP.     

The special location of p-H3 and p-CENP-A on heterochromatin during mitosis in MCF-7

CENP-A locates at nucleosome as histone H3-like proteins, and is phosphorylated during mitosis. We investigated the dynamic distribution of p-CENP-A to explore the details of its function. We found that p-CENP-A was phosphorylated at late prophase, and the signal of p-CENP-A arranged at equatorial plate along with nucleosomes at metaphase, but moved to midbody at later phase of mitosis. The phosphorylation modification of CENP-A shares some characters of H3, but has different temporal patterns during mitosis. Our results suggested that the CENP-A might have similar functions as H3, but with different patterns for their different binging materials. Dengwen Li and Ruming Liu had contributed equally to this paper.     

In vitro activity of human translation initiation factor eIF4B is not affected by phosphomimetic amino acid substitutions S422D and S422E

Eukaryotic translation initiation factor eIF4B is necessary for ribosomal scanning through structured mRNA leaders. In higher eukaryotes, eIF4B serves as a downstream effector of several signaling pathways. In response to mitogenic stimuli, eIF4B undergoes multiple phosphorylations which are thought to regulate its activity. Recently, Ser422 was identified as a predominant site for human eIF4B phosphorylation via several signaling pathways, and phosphomimetic amino acid substitutions S422D or S422E were shown to activate eIF4B in living cells. However, stimulatory role of these modifications has never been analyzed directly. Here, using both mammalian reconstituted translation initiation assay and complete cell-free translation system, we perform a comparison of recombinant eIF4B derivatives with the wild type recombinant protein, and do not find any difference in their activities. On the contrary, native eIF4B purified from HeLa cells reveals significantly higher activity in both assays. Thus, the effects of S422D and S422E substitutions on eIF4B activity in living cells observed previously either require some other protein modification(s), or may only be manifested in an intact cell. Our study raises the question on whether the phosphorylation of Ser422 is sufficient for eIF4B activation observed upon mitogenic stimulation.     

Prevalence of known mutations and a novel missense mutation (M694K) in the MEFV gene in a population from the Eastern Anatolia Region of Turkey

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and serositis. Mutations in the Mediterranean fever gene (MEFV) localized on the short arm of chromosome 16 cause FMF. Over 90 MEFV missense/nonsense mutations have been identified so far in FMF patients, mostly in the 10th exon of the gene.     

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F₀F₁ ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F₀F₁ complex is Mg²⁺-dependent and moderately stimulated by high concentrations of Ca²⁺ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F₀F₁ ATPase inhibitors that react with the membrane-bound F₀ complex. The assay of biochemical properties of the F₀F₁ ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F₀F₁ complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F₀F₁ ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.     

Regulation of an alternative sigma factor σI by a partner switching mechanism with an anti-sigma factor PrsI and an anti-anti-sigma factor ArsI in Streptomyces coelicolor A3(2)

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376-78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211-213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.