The arrestin-domain containing protein AdcA is a response element to stress.

BACKGROUND: Cell behaviour is tightly determined by sensing and integration of extracellular changes through membrane detectors such as receptors and transporters and activation of downstream signalling cascades. Arrestin proteins act as scaffolds at the plasma membrane and along the endocytic pathway, where they regulate the activity and the fate of some of these detectors. Members of the arrestin clan are widely present from unicellular to metazoa, with roles in signal transduction and metabolism. As a soil amoeba, Dictyostelium is frequently confronted with environmental changes likely to compromise survival. Here, we investigated whether the recently described arrestin-related protein AdcA is part of the cell response to stresses. RESULTS: Our data provide evidence that AdcA responds to a variety of stresses including hyperosmolarity by a transient phosphorylation. Analysis in different mutant backgrounds revealed that AdcA phosphorylation involves pathways other than the DokA and cGMP-dependent osmostress pathways, respectively known to regulate PKA and STATc, key actors in the cellular response to conditions of hyperosmolarity. Interestingly, however, both AdcA and STATc are sensitive to changes in the F-actin polymerization status, suggesting a common primary sensor/trigger and linking the stress-sensitive kinase responsive for AdcA phosphorylation to the actin cytoskeleton. We also show that STATc-dependent transcriptional activity is involved for the timely dephosphorylation of AdcA in cells under stress. CONCLUSION: Under osmotic stress, AdcA undergoes a phosphorylation-dephosphorylation cycle involving a stress-sensitive kinase and the transcription regulator STATc. This transient post-transcriptional modification may allow a regulation of AdcA function possibly to optimize the cellular stress response.

True arrestins and arrestin-fold proteins: a structure-based appraisal.

Arrestin-clan proteins are folded alike, a feature responsible for their recent grouping in a single clan. In human, it includes the well-characterized visual and ß-arrestins, the arrestin domain-containing proteins (ARRDCs), isoforms of the retromer subunit VPS26, and DSCR3, a protein involved in Down syndrome. A new arrestin-fold-predicted protein, RGP1, described here may join the clan. Unicellular organisms like the yeast Saccharomyces cerevisiae or the amoeba Dictyostelium discoideum harbor VPS26, DSCR3, and RGP1 isoforms as well as arrestin-related trafficking adaptors or ADCs, but true arrestins are missing. Functionally, members of the arrestin clan have generally a scaffolding role in various membrane protein trafficking events. Despite their similar structure, the mechanism of cargo recognition and internalization and the nature of recruited partners differ for the different members. Based on the recent literature, true arrestins (visual and ß-arrestins), ARRDCs, and yeast ARTS are the closest from a functional point of view.

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry.

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.

FYVE-dependent endosomal targeting of an arrestin-related protein in amoeba.

BACKGROUND: Visual and ß-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.

The arrestin fold: variations on a theme.

Endocytosis of ligand-activated plasma membrane receptors has been shown to contribute to the regulation of their downstream signaling. beta-arrestins interact with the phosphorylated tail of activated receptors and act as scaffolds for the recruitment of adaptor proteins and clathrin, that constitute the machinery used for receptor endocytosis. Visual- and beta-arrestins have a two-lobe, immunoglobulin-like, beta-strand sandwich structure. The recent resolution of the crystal structure of VPS26, one of the retromer subunits, unexpectedly evidences an arrestin fold in this protein, which is otherwise unrelated to arrestins. From a functional point of view, VPS26 is involved in the retrograde transport of the mannose 6-P receptor from the endosomes to the trans-Golgi network. In addition to the group of genuine arrestins and Vps26, mammalian cells harbor a vast repertoire of proteins that are related to arrestins on the basis of their PFAM Nter and Cter arrestin- domains, which are named Arrestin Domain- Containing proteins (ADCs). The biological role of ADC proteins is still poorly understood. The three subfamilies have been merged into an arrestin-related protein clan.This paper provides an overall analysis of arrestin clan proteins. The structures and functions of members of the subfamilies are reviewed in mammals and model organisms such as Drosophila, Caenorhabditis, Saccharomyces and Dictyostelium.

Necrotic cell death: From reversible mitochondrial uncoupling to irreversible lysosomal permeabilization.

Dictyostelium atg1- mutant cells provide an experimentally and genetically favorable model to study necrotic cell death (NCD) with no interference from apoptosis or autophagy. In such cells subjected to starvation and cAMP, induction by the differentiation-inducing factor DIF or by classical uncouplers led within minutes to mitochondrial uncoupling, which causally initiated NCD. We now report that (1) in this model, NCD included a mitochondrial-lysosomal cascade of events, (2) mitochondrial uncoupling and therefore initial stages of death showed reversibility for a surprisingly long time, (3) subsequent lysosomal permeabilization could be demonstrated using Lysosensor blue, acridin orange, Texas red-dextran and cathepsin B substrate, (4) this lysosomal permeabilization was irreversible, and (5) the presence of the uncoupler was required to maintain mitochondrial lesions but also to induce lysosomal lesions, suggesting that signaling from mitochondria to lysosomes must be sustained by the continuous presence of the uncoupler. These results further characterized the NCD pathway in this priviledged model, contributed to a definition of NCD at the lysosomal level, and suggested that in mammalian NCD even late reversibility attempts by removal of the inducer may be of therapeutic interest.

Mitochondrial carrier family: repertoire and peculiarities of the cellular slime mould Dictyostelium discoideum.

Proteins of the mitochondrial carrier family (MCF) mediate the transport of a large range of compounds, including metabolites and cofactors. They are localized mainly in the inner mitochondrial membrane, except for a few members found in the membranes of peroxisomes. Similarity searches among Dictyostelium discoideum protein sequences identified a total of 31 MCF members. All these are membrane proteins that possess three characteristic repeats of a domain of approximately 100 residues. Among them, three proteins have supplementary structural domains consisting of Ca(2+)-binding motifs made up of 2 or 4 EF-hand units localized on the N-terminal end, facing the mitochondrial intermembrane space. The nature of transported substrates is proposed on the basis of sequence comparison with orthologs characterized biochemically in other organisms, of phylogenetic analysis, and of the conservation of discriminating amino acid residues belonging to the substrate binding sites. Carriers have been grouped in subclasses based on their specificity for the transport of nucleotides, amino acids or keto acids. Furthermore, we have identified an iron carrier of the mitoferrin type, an inorganic phosphate carrier, and three carriers with similarity to uncoupler proteins. This study provides a focus for mitochondrial carrier analysis in Dictyostelium discoideum.

Purification techniques of subcellular compartments for analytical and preparative purposes.

In the following protocols, broken cells are the starting material of all downstream purifications of functional organelles or intact subcellular membranes. The choice of the breakage method has direct and deep repercussions on the quality of subsequent steps. Breaking vegetative amoebae by shear stress with a steel ball cell cracker preserves the integrity of subcellular organelles and in particular that of lysosomes, the rupture of which is very deleterious to further purifications. In this chapter, we propose purification schemes for plasma membrane, nuclei, mitochondria, and endocytic compartments. Plasma membranes are purified without any cell coating by partition between aqueous polymer phases. Nuclei and mitochondria are purified by differential centrifugations in adequate buffer conditions. Endosomes are magnetically isolated after feeding the cells with colloidal iron dextran and phagosomes by flotation on a sucrose gradient after feeding amoebae with latex beads. As analytical approaches, we propose procedures to label the plasma membrane and the endo-lysosomal compartments by biotinylation and to separate early and late compartments on a Percoll gradient.

Trafficking and developmental signaling: Alix at the crossroads.

Alix is a phylogenetically conserved protein that participates in mammals in programmed cell death in association with ALG-2, a penta-EF-hand calciprotein. It contains an N-terminal Bro1 domain, a coiled-coil region and a C-terminal proline-rich domain containing several SH3- and WW-binding sites that contribute to its scaffolding properties. Recent data showed that by virtue of its Bro1 domain, Alix is functionally associated to the ESCRT complexes involved in the biogenesis of the multivesicular body and sorting of transmembrane proteins within this specific endosomal compartment. In Dictyostelium, an alx null strain shows a markedly perturbed starvation-induced morphogenetic program while ALG-2 disruptants remain unaffected. This review summarizes Dictyostelium data on Alix and ALG-2 homologues and evaluates whether known functions of Alix in other organisms can account for the developmental arrest of the alx null mutant and how Dictyostelium studies can substantiate the current understanding of the function(s) of this versatile and conserved signaling molecule.

Specific host genes required for the killing of Klebsiella bacteria by phagocytes.

The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum.

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.

Dd-Alix, a conserved endosome-associated protein, controls Dictyostelium development.

We have characterized the Dictyostelium homolog of the mammalian protein Alix. Dd-Alix is encoded by a single gene and is expressed during vegetative growth and multicellular development. We showed that the alx null strain fails to complete its developmental program. Past the tight aggregate stage, morphogenesis is impaired, leading to markedly aberrant structures containing vacuolated and undifferentiated cells but no mature spores. The developmental defect is cell-autonomous as most cells remain of the PstB type even when mixed with wild-type cells. Complementation analysis with different Alix constructs allowed the identification of a 101-residue stretch containing a coiled-coil domain essential for Alix function. In addition, we showed that the protein associates in part with vesicular structures and that its distribution on a Percoll gradient overlaps that of the endocytic marker Vamp7. Dd-Alix also co-localizes with Dd-Vps32. In view of our data, and given the role of Vps32 proteins in membrane protein sorting and multivesicular body formation in yeast and mammals, we hypothesize that the developmental defects of the alx null strain result from abnormal trafficking of cell-surface receptors.

Artificial metalloenzymes: (strept)avidin as host for enantioselective hydrogenation by achiral biotinylated rhodium-diphosphine complexes.

We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium-diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin). The association constant between a prototypical cationic biotinylated rhodium-diphosphine catalyst precursor and the host proteins was determined at neutral pH: log K(a) = 7.7 for avidin (pI = 10.4) and log K(a) = 7.1 for streptavidin (pI = 6.4). It is shown that the optimal operating conditions for the enantioselective reduction are 5 bar at 30 degrees C with a 1% catalyst loading.

Developmental cell death in dictyostelium does not require paracaspase.

Apoptotic cell death often requires caspases. Caspases are part of a family of related molecules including also paracaspases and metacaspases. Are molecules of this family generally involved in cell death? More specifically, do non-apoptotic caspase-independent types of cell death require paracaspases or metacaspases? Dictyostelium discoideum lends itself well to answering these questions because 1) it undergoes non-apoptotic developmental cell death of a vacuolar autophagic type and 2) it bears neither caspase nor metacaspase genes and apparently only one paracaspase gene. This only paracaspase gene can be inactivated by homologous recombination. Paracaspase-null clones were thus obtained in each of four distinct Dictyostelium strains. These clones were tested in two systems, developmental stalk cell death in vivo and vacuolar autophagic cell death in a monolayer system mimicking developmental cell death. Compared with parent cells, all of the paracaspase-null cells showed unaltered cell death in both test systems. In addition, paracaspase inactivation led to no alteration in development or interaction with a range of bacteria. Thus, in Dictyostelium, vacuolar programmed cell death in development and in a monolayer model in vitro would seem not to require paracaspase. To our knowledge, this is the first instance of developmental programmed cell death shown to be independent of any caspase, paracaspase or metacaspase. These results have implications as to the relationship in evolution between cell death and the caspase family.

A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases.

The water soluble fluorescein-based ligand 1 forms a non-fluorescent complex with Cu(2+). This complex serves as a fluorescent sensor for amino acids in the 10(-3) M concentration range. Since the signal response is very fast, the sensor can be used to detect the hydrolytic activity of various proteases (trypsin, chymotrypsin, subtilisin) on bovine serum albumin as a whole protein substrate, and more generally to follow reactions releasing or removing free amino acids, in real time.

Fluorogenic stereochemical probes for transaldolases.

Transaldolase catalyzes the transfer of dihydroxyacetone from, for example, fructose 6-phosphate to erythrose 4-phosphate. As a potential probe for assaying fluorescent transaldolase, 6-O-coumarinyl-fructose (1) was prepared in six steps from D-fructose. The corresponding 6-O-coumarinyl-5-deoxy derivative 2 was prepared stereoselectively from acrolein and tert-butyl acetate by a chemoenzymatic route involving Amano PS lipase for the kinetic resolution of tert-butyl 3-hydroxypent-4-enoate (7) and E. coli transketolase for assembly of the final product. The corresponding stereoisomer related to D-tagatose was obtained by a chemical synthesis starting from D-ribose. Indeed, transaldolases catalyze the retro-aldolization of substrate 1 to give dihydroxyacetone and 3-O-coumarinyl-glyceraldehyde. The latter primary product undergoes a beta-elimination in the presence of bovine serum albumin (BSA) to give the strongly fluorescent product umbelliferone. A similar reaction is obtained with the 5-deoxy analogue 2, but there is almost no reaction with its stereoisomer 3. The stereoselectivity of transaldolases can be readily measured by the relative rates of fluorescence development in the presence of the latter pair of diastereomeric substrates.

Parallel solid-phase synthesis of trisubstituted triazinobenzimidazolediones.

An efficient method for the solid-phase synthesis of trisubstituted [1,3,5]triazino[1,2-a]benzimidazole-2,4(3H,10H)-diones from resin-bound amino acids is described. N-acylation of the primary amine of a resin-bound amino acid with 4-fluoro-3-nitrobenzoic acid, followed by displacement of the fluoro group and reduction of the nitro group, generated a resin-bound o-dianilino derivative. The dianilino compound was treated with cyanogen bromide to generate the corresponding iminobenzimidazole, which, following treatment with N-(chlorocarbonyl)isocyanate, afforded the resin-bound triazinodione derivative. Alkylation of the triazinodione compound with an alkyl halide yielded, following cleavage of the solid-support, the trisubstituted [1,3,5]triazino[1,2-a]benzimidazole-2,4(3H,10H)-dione.

Biochemical characterization of two analogues of the apoptosis-linked gene 2 protein in Dictyostelium discoideum and interaction with a physiological partner in mammals, murine Alix.

Two homologues, Dd-ALG-2a and Dd-ALG-2b, of the mammalian calcium-binding protein ALG-2 (apoptosis-linked gene 2) have been characterized in the cellular slime mold Dictyostelium discoideum. Fluorescence titrations showed that both proteins bind calcium ions with affinities (Ca2+)(0.5) of 30 and 450 microm, respectively, at sites specific to calcium. Calcium ion binding resulted in changes of conformation associated with the unmasking of hydrophobic regions of the proteins. Surface plasmon resonance analysis showed that Dd-ALG-2a homodimers formed (K(D) of 1 microm) at calcium ion concentrations similar to those necessary for Ca2+-induced conformational changes. Deletion of the hydrophobic N-terminal sequence or EF-hand 5 of Dd-ALG-2a prevented dimerization. The Dd-ALG-2b homodimer was not detected, and the Dd-ALG-2a/2b heterodimer formed only when Dd-ALG-2b was the immobilized partner. Murine Alix formed a heterodimer (K(D) = 0.6 microm) with Dd-ALG-2a but not with Dd-ALG-2b, and the interaction strictly depended upon calcium ions. The DeltaNter construct of Dd-ALG-2a lost its interaction capacity with mouse Alix. The genes encoding both proteins, Dd-alg-2a and -2b, were expressed in growing cells. The levels of mRNA were at a maximum during aggregation (4-8 h) and decreased rapidly thereafter. In contrast, the levels of proteins remained fairly stable. Dd-ALG-2a and Dd-ALG-2b were found to be dispensable for growth and development, based on the finding that single Dd-alg2a- or Dd-alg-2b- and double Dd-alg2a-/Dd-alg-2b- mutant cell lines showed normal growth in axenic medium or on bacterial lawns and exhibited unaltered development.