Design, synthesis, and evaluation of proline based melanocortin receptor ligands

A series of proline based melanocortin ligands has been developed on the basis of initial piperazine leads by using a more conformationally rigid scaffold. A number of these novel ligands showed significant binding affinity for MC3 and MC4 receptors.     

PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis

Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.     

Mitochondrial haplotype diversity in the tortoise species <Emphasis Type="Italic">Testudo graeca</Emphasis> from North Africa and the Middle East

Background  

To help conservation programs of the endangered spur-thighed tortoise and to gain better insight into its systematics, genetic variation and evolution in the tortoise species Testudo graeca (Testudines: Testudinidae) was investigated by sequence analysis of a 394-nucleotide fragment of the mitochondrial 12S rRNA gene for 158 tortoise specimens belonging to the subspecies Testudo graeca graeca, Testudo graeca ibera, Testudo graeca terrestris, and a newly recognized subspecies Testudo graeca whitei. A 411-nucleotide fragment of the mitochondrial D-loop was additionally sequenced for a subset of 22 T. graeca, chosen because of their 12S gene haplotype and/or geographical origin.     

Xenopus as a model organism in developmental chemical genetic screens

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.     

p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association

Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.     

The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex

Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.     

Cytoplasmic targeting signals in transmembrane invariant surface glycoproteins of trypanosomes

Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family.     

Redox-dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide reductase revealed by cyanide binding studies

The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron.     

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.     

Evaluation of an innate immune reaction to parasites in earthworms

Encapsulation is an essential process of the invertebrate immune system and includes the prophenoloxidase (proPO) cascade. We present an assay for evaluating this immune response, now newly adapted to earthworms. Coelomic fluid is withdrawn and coelomocytes are stained with l-Dopa. We studied assay repeatability and the correlation between number of PO-active cells and infection level of the parasitic protozoan Monocystis sp. in the earthworm Lumbricus terrestris. Our study showed high assay repeatability although the expected negative relationship between PO-active coelomocytes and parasite load was not observed; yet a suggestion toward a positive relationship was detected. This finding is contrary to previous assumptions that presume coelomocyte concentrations to be the independent variable determining parasite load.