Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling

Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.     

Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.     

Chemical complementation: small-molecule-based genetic selection in yeast

Protein and metabolic engineering would greatly benefit from a general system linking the presence of a small molecule to the power of genetic selection. We use nuclear receptors to link the survival of Saccharomyces cerevisiae to the presence of small molecules through genetic selection, extending classical genetic complementation to a new "chemical complementation." In this system the Gal4 DNA-binding domain is fused to ligand-binding domains from two nuclear receptors, expressed in the strain PJ69-4A, and grown on plates containing known ligands for the receptors. Yeast survive on selective plates only in the presence of a nuclear receptor and the corresponding ligand. Mutagenesis can increase the sensitivity of chemical complementation. This system may be extended to engineer nuclear receptors for practically any small molecule through directed evolution coupled to genetic selection, and for performing metabolic engineering in yeast.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.     

Enhanced selection for MHC diversity in social tuco-tucos

To explore the effects of behavior and demography on balancing selection at major histocompatibility complex (MHC) loci, we examined allelic diversity at exon 2 of the MHC class II DQbeta locus in a social and a solitary species of tuco-tuco (Rodentia: Ctenomyidae: Ctenomys), both of which occur in the same valley in southwestern Argentina. By comparing patterns of diversity at this MHC gene to the diversity evident at fifteen microsatellite loci, we demonstrate that balancing selection at the DQbeta locus is enhanced in the social species compared to its solitary congener. These findings have intriguing implications for the role of behavioral and demographic parameters in maintaining diversity at MHC loci.     

MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake

The Staphylococcus aureus DtxR-like protein, MntR, controls expression of the mntABC and mntH genes, which encode putative manganese transporters. Mutation of mntABC produced a growth defect in metal-depleted medium and increased sensitivity to intracellularly generated superoxide radicals. These phenotypes resulted from diminished uptake of manganese and were rescued by the addition of excess Mn(II). Resistance to superoxide was incompletely rescued by Mn(II) for STE035 (mntA mntH), and the strain had reduced virulence in a murine abscess model of infection. Expression of mntABC was repressed by Mn(II) in an MntR-dependent manner, which contrasts with the expression of mntH that was not repressed in elevated Mn(II) and was decreased in an mntR mutant. This demonstrates that MntR acts as a negative and positive regulator of these loci respectively. PerR, the peroxide resistance regulon repressor, acts with MntR to control the expression of mntABC and manganese uptake. The expression of the PerR-regulated genes, katA (catalase), ftn (ferritin) and fur (ferric uptake regulator), was diminished in STE031 (mntR) when grown in excess Mn(II). Therefore, the control of Mn(II)-regulated members of the PerR regulon and the Fur protein is modulated by MntR through its control of Mn(II) uptake. The co-ordinated regulation of metal ion homeostasis and oxidative stress resistance via the regulators MntR, PerR and Fur of S. aureus is discussed.     

Clathrin-protein interactions

There is a complex network of protein–protein and protein–lipid interactions that underlie clathrin-mediated vesicular traffic in all compartmentalized cells from yeast to man. Major progress has been made in the determination of the three-dimensional structures of many of the components. Recently, there has been an explosion in the identification and characterization of clathrin binding partners. This review integrates the structural and biochemical information that is currently available to present a unified view of how many clathrin binding partners interact with clathrin.     

Jasmonate-induced lipid peroxidation in barley leaves initiated by distinct 13-LOX forms of chloroplasts

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [V?r?s et al., Eur. J. Biochem. 251 (1998), 36-44], two full-length cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonate-treated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenase-derived products in the stroma and in the envelope. These data revealed jasmonate-induced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.