Substrate-based design of reversible Pin1 inhibitors

Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m) < 5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i) > 1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.     

Thermodynamic characterization of the interaction of human cyclophilin 18 with cyclosporin A

Isothermal titration calorimetry (ITC) was used to investigate thermodynamic parameters of the cyclosporin A (CsA)-cyclophilin 18 (hCyp18) association reaction. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy of binding) of the CsA/hCyp18 complexation. All but two methods described in the literature underestimate the affinity to hCyp18 of CsA. We found that the association constant (1.1·10⁸ M⁻¹ at 10 °C) of CsA to hCyp18 is in close agreement with the reciprocal of the reported inhibitory constant of the peptidylprolyl cis/trans isomerase activity of hCyp18. Interpretation of the thermodynamic parameters in buffered solution of water, 30% glycerol and D₂O leads to the conclusion that the highly specific binding of CsA to hCyp18 is mainly mediated through hydrogen bonding and to a lesser degree through hydrophobic interaction. Furthermore, the pH dependence of the association constant was determined and analyzed according to a single proton linkage model, resulting in a pK_a value of 5.7 in free hCyp18 and below 4.5 in the CsA complexed form. Titration experiments using different single component buffers possessing different heats of ionization allowed us to estimate that statistically half a proton is transferred upon CsA binding from the binding interface of hCyp18 to the buffer at pH 5.5. No proton transfer was detected at pH 7.5. The thermodynamic results are discussed in relation to the published X-ray and NMR structure of the free and CsA complexed hCyp18.     

SH2-B is required for growth hormone-induced actin reorganization

The Src homology-2 (SH2) domain-containing protein SH2-Bbeta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-Bbeta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-Bbeta present at the plasma membrane and in the cytosol. SH2-Bbeta colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-Bbeta is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-Bbeta in 3T3-F442A cells and assayed for GH- and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-Bbeta enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-Bbeta (R555E) and truncation mutant DeltaC555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant DeltaN504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. DeltaN504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-Bbeta is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-Bbeta as a stimulator of JAK2 kinase activity.     

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.     

Lateral organs in sedentary polychaetes (Annelida) – Ultrastructure and phylogenetic significance of an insufficiently known sense organ

Lateral organs are sense organs visible as densely ciliated pits or papillae between the noto‐ and the neuropodia in certain taxa of sedentary polychaetes. Ultrastructural studies in about 10 species of the following taxa Maldanidae, Opheliidae, Orbiniidae, Paraonidae, Magelonidae, Spionidae, Poecilochaetidae and Terebellidae have been designed to evaluate whether these organs are homologous among polychaetes. In spite of great external diversity, the investigations revealed an overall ultrastructural similarity. Differences between species investigated mainly concern the size of the organs as well as the number and arrangement of cells. The organs comprise supportive cells and uniciliated penetrative sensory cells. Their dendrites are closely arranged and thus their cilia may resemble multiciliated cells. There are two types of sensory cells: one type possesses no or mainly thin microvilli of which usually only a few reach the cuticular surface, and in the other type the cilium is consistently surrounded by 10 strong microvilli, which form a pore‐like opening in the cuticle. Further differences occur in the structure of the rootlet system. Basally, a retractor muscle attaches to the organ. The systematic significance of these organs within Annelida is discussed with respect to the conflicting phylogenetic hypotheses explaining the relationships of annelid taxa.     

A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments

Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus “Scalindua” group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem.     

Musculature in polychaetes: comparison of Myrianida prolifera (Syllidae) and Sphaerodoropsis sp. (Sphaerodoridae)

Abstract. The relationship of the polychaete taxa Syllidae and Sphaerodoridae within Phyllodocida is still unresolved: phylogenetic analyses either show them as sister groups or more widely separated. The present article aims to provide information about the structure of the muscular system that could be essential for understanding their relationship. A crucial point is whether the body wall contains circular muscles, which has recently been shown to be absent in more taxa than previously known. The F-actin filaments in members of Myrianida prolifera (Syllidae) and Sphaerodoropsis sp. (Sphaerodoridae) were labeled with phalloidin and their three-dimensional relationships reconstructed by means of confocal laser scanning microscopy. Among the noteworthy differences that emerged between the species are (1) members of M. prolifera possess four, those of Sphaerodoropsis sp. eight, longitudinal muscle strands; (2) the body wall in M. prolifera contains transverse fibers in a typical, supralongitudinal position, while in Sphaerodoropsis sp., corresponding fibers lie beneath the longitudinal strands; (3) pro- and peristomium in M. prolifera have no distinct F-actin fibers, while five longitudinal pairs and three single transverse muscular fibers shape the anterior end in Sphaerodoropsis sp.; (4) the proventricle of M. prolifera comprises primarily radial muscle fibers arranged in distinct rows, while in Sphaerodoropsis sp. the axial proboscis consists of longitudinal and circular fibers and radial fibers are lacking; (5) in M. prolifera, the proximal and distal sections of the two anteriormost pairs of dorsal cirri possess longitudinal myofilaments, which are separate from the body wall musculature; by contrast, all appendages in Sphaerodoropsis sp. do not; (6) both species have bracing muscles: in M. prolifera they are positioned above the longitudinal fibers, whereas in Sphaerodoropsis sp. they are uniquely positioned between longitudinal and sublongitudinal transverse fibers. These results do not support a sister-group relationship of Syllidae and Sphaerodoridae. In addition, Sphaerodoropsis sp. is yet another example in the list of polychaetes lacking typical circular muscles in the body wall.