Occurrence of articulins and epiplasmins in protists

The cortex of ciliates. dinoflagellates, and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm in the flagellate Euglena gracilis and the ciliate Pseudomicrothorax dubius. The hallmark of articulins is a core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. The VPV-motif repeats are 12 residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. Using molecular and immunological tools we show that articulins are also present in the dinoflagellate Amphidinium carterae and the ciliates Paramecium tetraurelia and Paramecium caudatum, Tetrahymena pyriformis, and Euplotes aediculatus. Our analysis further shows that epiplasmins, a group of epiplasmic proteins first characterized in Paramecium, are also present in all these species. Moreover, we present evidence that epiplasmins and articulins represent two distinct classes of cytoskeletal proteins.     

A gene fusion event in the evolution of aminoacyl-tRNA synthetases

The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life. In bacteria and archaea, distinct genes encode the two proteins. In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein. The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases. Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl-tRNA synthetases. In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively. Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units.     

MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae

The gene MUS81 (Methyl methansulfonate, UV sensitive) was identified as clone 81 in a two-hybrid screen using the Saccharomyces cerevisiae Rad54 protein as a bait. It encodes a novel protein with a predicted molecular mass of 72,316 (632 amino acids) and contains two helix-hairpin-helix motifs, which are found in many proteins involved in DNA metabolism in bacteria, yeast, and mammals. Mus81p also shares homology with motifs found in the XPF endonuclease superfamily. Deletion of MUS81 caused a recessive methyl methansulfonate- and UV-sensitive phenotype. However, mus81Δ cells were not significantly more sensitive than wild-type to γ-radiation or double-strand breaks induced by HO endonuclease. Double mutant analysis suggests that Rad54p and Mus81p act in one pathway for the repair of, or tolerance to, UV-induced DNA damage. A complex containing Mus81p and Rad54p was identified in immunoprecipitation experiments. Deletion of MUS81 virtually eliminated sporulation in one strain background and reduced sporulation and spore viability in another. Potential homologs of Mus81p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans and Arabidopsis thaliana. We hypothesize that Mus81p plays a role in the recognition and/or processing of certain types of DNA damage (caused by UV and MMS) during repair or tolerance processes involving the recombinational repair pathway. Received: 9 December 1999 / Accepted: 24 February 2000     

Ligand-receptor interactions as controlled by wild-type and mutant Thr(370)Lys alpha2B-adrenoceptor-Galpha15 fusion proteins

Fusion proteins were constructed between either a wild-type or mutant Thr370Lys alpha2B-adrenoceptor (alpha2B AR) and a mouse Galpha15 protein to analyze ligand-receptor interactions at a receptor/Galpha15 protein density ratio of 1. Activation of the wild-type alpha2B AR-Galpha15 fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50 = 7.37+/-0.13) increase in the intracellular Ca2+ concentration, which could be antagonized by RX 811059 (pK(B) = 7.55+/-0.15). Whereas d-medetomidine and oxymetazoline were as efficacious agonists as (-)-adrenaline, the following ligands displayed partial agonist properties: BRL 44408 < atipamezole < clonidine < UK 14304 < BHT 920. A comparison with the mutant Thr370Lys alpha2B AR-Galpha15 fusion protein displayed similar Ca2+ kinetics and a ligand-mediated receptor activation profile characterized by higher potencies and greater maximal Ca2+ responses for the ligands being investigated, including the putative antagonists dexefaroxan and idazoxan. RX 811059 and RX 821002 remained silent. Similar conclusions could be made on enhancement of the ligands' intrinsic activities by coexpression of the mutant Thr370Lys alpha2B AR with either a Galpha15 or Galphao Cys351Ile protein. The Thr370Lys alpha2B AR-Galpha protein interactions may modify the tertiary structure of the mutant receptor in such a way that some putative alpha2 AR antagonists are capable of stabilizing an active receptor conformation, thereby generating positive efficacy.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 20 (S 1197). Compound 20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure–activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the -amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of 20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

A biomimetic strategy in the synthesis and fragmentation of cyclic protein.

This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine-knot motif with three disulfide bonds and an end-to-end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiol-thiolactone exchanges to arrive at an alpha-amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S,N-acyl migration to form the cyclic protein. A two-step disulfide formation strategy is employed for obtaining the desired disulfide-paired products. Partial acid hydrolysis through intramolecular acyl transfer of X-Ser, X-Thr, Asp-X, and Glu-X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine-knot motif of 1-4, 2-5, and 3-6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and characterization of large end-to-end cyclic peptides and small proteins.     

U-richness is a defining feature of plant introns and may function as an intron recognition signal in maize

Using a large set of plant gene sequences we compared individual introns to their flanking exons. Both Zea mays and Arabidopsis thaliana introns are U-rich but display no apparent bias for A. We identified fifteen 11-mer U-rich motifs as frequent elements of maize introns, and these are virtually absent from exons. By mutagenesis, we show that the single U-rich motif in the Bronze2 intron of maize plays a key role in intron processing in vivo.     

Synthesis and conformation of an analog of the helix‐loop‐helix domain of the Id1 protein containing the O‐acyl iso‐prolyl‐seryl switch motif

Synthetic peptides reproducing the helix‐loop‐helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self‐association. Recently, we have shown that the replacement of the dipeptide Val‐Ser at the loop–helix‐2 junction with the corresponding O‐acyl iso‐dipeptide leads to a completely unfolded state that only refolds after intramolecular O → N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro‐Ser motif at the helix‐1–loop junction with the corresponding O‐acyl iso‐dipeptide. This analog has been successfully synthesized by solid‐phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O‐acyl iso‐peptide before its conversion into the native form by O → N acyl shift. These results show that the loop–helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O‐acyl iso‐Pro‐Ser/Thr units are synthetically accessible by Fmoc chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.