eIF2α kinases control chalone production in Dictyostelium discoideum

Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α.     

The p21-Activated Kinase (PAK) Family Member PakD Is Required for Chemorepulsion and Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK) family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass) on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.     

A Dictyostelium Secreted Factor Requires a PTEN-Like Phosphatase to Slow Proliferation and Induce Chemorepulsion

In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN¯ cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN¯ cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN¯ cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.     

The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA⁻ cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA⁻ cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.Physiological processes that define and maintain the sizes of tissues are poorly understood. Although a number of characterized gene products negatively regulate the sizes of tissues (21, 23), the mechanism by which the activities of such gene products are controlled is unclear. One potential mechanism for tissue size regulation consists of tissue-specific autocrine signals that inhibit proliferation in a concentration-dependent manner (18). Since the extracellular concentration of such factors increases as a function of cell density and/or cell number, the proliferation-inhibiting function of these factors can limit tissue size. Considerable evidence for such factors has been reported. For instance, full hepatectomy in one of two rats with conjoined circulatory systems stimulated proliferation in the intact liver of the conjoined rat, suggesting the existence of a systemic factor produced by the liver that inhibits the proliferation of hepatocytes (16). However, only a small number of factors with analogous functional roles, such as myostatin, which regulates skeletal muscle size (30), and Gdf11, which negatively regulates neurogenesis in the olfactory epithelium (38), have been identified. The mechanisms by which such signals inhibit proliferation are not well understood. As such autocrine signals may serve to limit tumor growth (14, 20), elucidation of the identities of such factors and their associated signal transduction pathways may yield novel cancer therapies.We have identified two such autocrine proliferation-repressing signals in the social amoeba Dictyostelium discoideum, a genetically and biochemically tractable model organism. The proteins AprA and CfaD are secreted by Dictyostelium and inhibit the proliferation of Dictyostelium cells in a concentration-dependent manner (4, 12). Cells in which the genes encoding either AprA or CfaD have been disrupted by homologous recombination proliferate rapidly, and cells overexpressing AprA or CfaD proliferate slowly (4, 11). Adding recombinant AprA (rAprA) or recombinant CfaD (rCfaD) to cells slows proliferation, demonstrating that these proteins function as extracellular signals (4, 12). In addition to exhibiting rapid proliferation, aprA⁻ and cfaD⁻ cells exhibit a multinucleate phenotype, strongly suggesting that AprA and CfaD are negative regulators of mitosis (4, 11). aprA⁻ cells are insensitive to the proliferation-inhibiting effects of CfaD (12), and cfaD⁻ cells are insensitive to AprA (4), indicating the necessity of both genes for proliferation inhibition and suggesting a common proliferation-inhibiting mechanism. The G protein complex subunits Gα8, Gα9, and Gβ are necessary for proliferation inhibition by AprA, and the addition of recombinant AprA to purified cell membranes increases binding of GTP to wild-type and gα9⁻ cell membranes but not gα8⁻ or gβ⁻ membranes, indicating that AprA activates a proliferation-inhibiting signal transduction pathway of which Gα8 and Gβ are components (5). The signal transduction pathway downstream of Gα8 and the associated mechanism of proliferation inhibition are unknown.Although the selective forces that have maintained functional autocrine proliferation inhibitors in proliferating Dictyostelium cells are unclear, AprA and CfaD may provide an advantage during the multicellular portion of the Dictyostelium life cycle. Upon starvation, Dictyostelium cells secrete pulses of the chemoattractant cyclic AMP, leading to cells streaming toward aggregation centers (15, 27). This process causes the formation of multicellular groups regulated in size by a secreted protein complex that stimulates stream breakup (9, 10). These groups develop into multicellular fruiting body structures composed of a mass of stress-resistant spores supported by an approximately 1-mm-high stalk (24). While the stalk cells inevitably die in an act of apparent altruism (31), the presence of nutrients stimulates spore germination and a continuation of proliferation (13). Following development, aprA⁻ and cfaD⁻ cells form fewer viable spores than the wild type (4, 11), suggesting that AprA and CfaD increase the fitness of Dictyostelium during development.Like aprA⁻ and cfaD⁻ cells, Dictyostelium cells lacking the ROCO family kinase QkgA have an abnormally rapid proliferation (1). The ROCO protein family is widely conserved and is defined by the presence of a Ras of complex protein (Roc) domain followed by a C terminus of Roc (Cor) domain, which mediates homodimerization (19). In eukaryotes, these domains are commonly followed C terminally by a kinase domain with similarity to the tyrosine kinase-like (TKL) group of kinases (3, 26, 29). In Dictyostelium, other ROCO proteins function in cyclic GMP signaling (8, 35) and cytokinesis (2), and a total of 11 predicted ROCO proteins are present in the genome, 10 of which, including QkgA, encode kinase domains predicted to be catalytically active (17). The human genome encodes two ROCO kinases, which are expressed in a wide range of tissues (25, 40). Little is known regarding the physiological functions of these proteins, although the ROCO protein LRRK2 is implicated in a dominantly inherited form of Parkinson''s disease (40) and negatively regulates neurite growth in rat cortical cultures (28).In this report, we show that, like aprA⁻ and cfaD⁻ cells, qkgA⁻ cells proliferate to a higher cell density than the wild type and tend to be multinucleate. Additionally, we show that qkgA⁻ cells are insensitive to exogenous AprA and CfaD, indicating that QkgA is required for AprA and CfaD signal transduction.     

A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum

Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA⁻ cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA⁻ cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA⁻ cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA⁻ cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA⁻ cells. Similar to aprA⁻ cells, rblA⁻ cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.     

A secreted factor represses cell proliferation in Dictyostelium

Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that inhibits the proliferation of wild-type and aprA- cells; this activity is not secreted by aprA- cells. AprA purified by immunoprecipitation also slows the proliferation of wild-type and aprA- cells. Compared with wild type, there is a higher percentage of multinucleate cells in the aprA- population, and when starved, aprA- cells form abnormal structures that contain fewer spores. AprA may thus decrease the number of multinucleate cells and increase spore production. Together, the data suggest that AprA functions as part of a Dictyostelium chalone.     

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

Background  

ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.     

Functional similarities between the dictyostelium protein AprA and the human protein dipeptidyl‐peptidase IV

Autocrine proliferation repressor protein A (AprA) is a protein secreted by Dictyostelium discoideum cells. Although there is very little sequence similarity between AprA and any human protein, AprA has a predicted structural similarity to the human protein dipeptidyl peptidase IV (DPPIV). AprA is a chemorepellent for Dictyostelium cells, and DPPIV is a chemorepellent for neutrophils. This led us to investigate if AprA and DPPIV have additional functional similarities. We find that like AprA, DPPIV is a chemorepellent for, and inhibits the proliferation of, D. discoideum cells, and that AprA binds some DPPIV binding partners such as fibronectin. Conversely, rAprA has DPPIV‐like protease activity. These results indicate a functional similarity between two eukaryotic chemorepellent proteins with very little sequence similarity, and emphasize the usefulness of using a predicted protein structure to search a protein structure database, in addition to searching for proteins with similar sequences.     

Exocytosis and protein secretion in <Emphasis Type="Italic">Trypanosoma</Emphasis>

Background  

Human African trypanosomiasis is a lethal disease caused by the extracellular parasite Trypanosoma brucei. The proteins secreted by T. brucei inhibit the maturation of dendritic cells and their ability to induce lymphocytic allogenic responses. To better understand the pathogenic process, we combined different approaches to characterize these secreted proteins.     

Encapsulated <Emphasis Type="Italic">Escherichia coli</Emphasis> in alginate beads capable of secreting a heterologous pectin lyase

Background  

Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.     

Functional and structural characteristics of anticancer peptide Pep27 analogues

Background  

A secreted peptide Pep27 initiates the cell death program in S. pneumoniae through signal transduction. This study was undertaken to evaluate the relation between the structure and cytotoxic activity of Pep27 and its analogues on cancer cells.     

Characterization of the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> surface-associated AP65 and binding domain interacting with trichomonads and host cells

Background  

AP65 is a prominent adhesin of Trichomonas vaginalis that mediates binding of parasites to host vaginal epithelial cells (VECs). AP65 with no secretion signal sequence, membrane targeting peptide, and anchoring motif was recently found to be secreted.     

<Emphasis Type="Italic">Bacillus cereus</Emphasis> cytotoxins Hbl,Nhe and CytK are secreted via the Sec translocation pathway

Background  

Bacillus cereus and the closely related Bacillus thuringiensis are Gram positive opportunistic pathogens that may cause food poisoning, and the three secreted pore-forming cytotoxins Hbl, Nhe and CytK have been implicated as the causative agents of diarrhoeal disease. It has been proposed that the Hbl toxin is secreted using the flagellar export apparatus (FEA) despite the presence of Sec-type signal peptides. As protein secretion is of key importance in virulence of a microorganism, the mechanisms by which these toxins are secreted were further investigated.     

Lbx2 regulates formation of myofibrils

Background  

Skeletal muscle differentiation requires assembly of contractile proteins into organized myofibrils. The Drosophila ladybird homeobox gene (lad) functions in founder cells of the segmental border muscle to promote myoblast fusion and muscle shaping. Tetrapods have two homologous genes (Lbx). Lbx1 functions in migration and/or proliferation of hypaxial myoblasts, whereas the function of Lbx2 is poorly understood.     

Resistance to cisplatin does not affect sensitivity of human ovarian cancer cell lines to mifepristone cytotoxicity

Background  

The prototypical antiprogestin mifepristone exhibits potent growth inhibition activity towards ovarian cancer cells in vitro and in vivo. The aim of this research was to establish whether mifepristone is capable of inhibiting cell proliferation and inducing apoptotic cell death regardless of the degree of sensitivity ovarian cancer cells exhibit to cisplatin.     

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development

Background  

Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.     

Experimental study of the function of the excreted/secreted <Emphasis Type="Italic">Leishmania</Emphasis>LmSIR2 protein by heterologous expression in eukaryotic cell line

Background  

In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells.