Protein 14-3-3zeta binds to protein phosphatase PP1gamma2 in bovine epididymal spermatozoa

The protein phosphatase PP1gamma2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1gamma2 is phosphorylated and that the amount of phosphorylated PP1gamma2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439-447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1gamma2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species-bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1gamma2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1gamma2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1gamma2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1gamma2 is one of its binding partners.     

Bacterial P450-catalyzed polyketide hydroxylation on a microfluidic platform

The incorporation of a multicomponent, cofactor-dependant P450 into a microfluidic biochip is demonstrated. The PikC hydroxylase Streptomyces venezuelae was incorporated into a PDMS-based microfluidic channel. The enzyme was immobilized to Ni-NTA agarose beads via in situ attachment following the addition of the beads to the microchannel. The enzyme loading was approximately 6 microg per mg of beads resulting in a microchannel loading of 10.7 mg/mL. This high enzyme loading enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin in about equal amounts with a conversion of >90% at a flow rate of 70 nL/min. This high reactivity allowed rapid hydroxylation reactions to be performed with short residence times, which is critical for complex enzymes with limited inherent stability.     

Mutational analysis of the fruA promoter region demonstrates that C-Box and 5-base-pair elements are important for expression of an essential developmental gene of Myxococcus xanthus

Myxococcus xanthus uses extracellular signals during development to regulate gene expression. C-signaling regulates the expression of many genes induced after 6 h into development. FruA is a protein that is necessary for cells to respond to C-signaling, but expression of the fruA gene does not depend on C-signaling. Yet the fruA promoter region has a C box and a 5-bp element, similar to the promoter regions of several C-signal-dependent genes, where these sequences are crucial. Here, we show that the C box and 5-bp elements are important for expression of fruA, demonstrating for the first time that these sequences play a role in the expression of a gene that does not depend on C-signaling and is required for M. xanthus development.     

Mutations in Saccharomyces cerevisiae iron-sulfur cluster assembly genes and oxidative stress relevant to Cu,Zn superoxide dismutase

Saccharomyces cerevisiae lacking Cu,Zn superoxide dismutase (SOD1) show several metabolic defects including aerobic blockages in methionine and lysine biosynthesis. We have previously shown that mutations in genes implicated in the formation of iron-sulfur clusters, designated seo (suppressors of endogenous oxidation), reverse the oxygen-dependent methionine and lysine auxotrophies of a sod1Delta strain. We now report the surprising finding that seo mutants do not reduce oxidative damage as shown by the lack of reduction of EPR-detectable "free" iron, which is characteristic of sod1Delta mutants. In fact, they exhibit increased oxidative damage as evidenced by increased accumulation of protein carbonyls. The seo class of mutants overaccumulates mitochondrial iron, and this iron accumulation is critical for suppression of the sod1Delta biosynthetic defects. Blocking overaccumulation of mitochondrial iron abolished the ability of the seo mutants to suppress the sod1Delta auxotrophies. By contrast, increasing the mitochondrial iron content of sod1Delta yeast using high copy MMT1, which encodes a mitochondrial iron transporter, was sufficient to mimic the seo mutants. Our studies indicated that suppression of the sod1Delta methionine auxotrophy was dependent on the pentose phosphate pathway, which is a major source of NADPH production. By comparison, the sod1Delta lysine auxotrophy appears to be reversed in the seo mutants by increased expression of genes in the lysine biosynthetic pathway, perhaps through sensing of mitochondrial damage by the retrograde response.     

CD80 binding polyproline helical peptide inhibits T cell activation

The critical role played by the CD28/CD152-CD80/CD86 costimulatory molecules in mediating T cell activation and suppression provides attractive targets for therapeutic strategies. CD28 and CD152 share a conserved polyproline motif in the ligand-binding region. Similar proline-rich regions in globular domains preferentially adopt a polyproline type II (PP) helical conformation and are involved in transient (II)protein-protein interactions. Interestingly, in the human CD80-CD152 complex, Pro(102) of CD152 restricts the preceding proline to PP(II) helix in the binding orientation in relation to the shallow binding pocket of CD80. Peptide agents derived from binding sites of receptors that mimic the bioactive conformation have been shown to block receptor-ligand interactions. Contact preferences of the interface amino acids at the protein-protein interaction sites and the propensity of each residue to form PP(II) helix were integrated in the design of novel peptide agents referred to as CD80 competitive antagonist peptides. Structural and functional studies suggest potential therapeutic value for select CD80 competitive antagonist peptides.     

Pharmacological analysis of ovarial patency in Heliothis virescens

The insect oocyte sequesters nutritive proteins during patency, which is facilitated as a result of intercellular spaces occurring between follicular epithelial cells under the influence of juvenile hormone (JH). Patency was analyzed in the moth, Heliothis virescens, using a pharmacological approach, in which we used different JH homologues and chemicals that specifically target elements of two second-messenger pathways in vertebrates, the cAMP-dependent and inositol triphosphate/diacylglycerol signaling pathways. JH I and JH III evoked dose-dependent patency in H. virescens oocyte follicles, which was suppressed by the Na/K-ATPase inhibitor, ouabain. Patency was observed in follicular epithelial cells treated with either protein kinase C activator, PDBu, or protein kinase A activator, 8-Br-cAMP, by itself. The protein kinase C inhibitor, H-7, preferentially suppressed patency evoked by JH III, whereas the protein kinase A inhibitor, H89, preferentially suppressed that evoked by JH I. Additionally, patency was triggered by the adenylate cyclase activator, NKH 477, or peptide Gs-protein activator, cholera toxin, alone. Patency evoked by JH I was suppressed by the adenylate cyclase inhibitor, SQ 22,536, and GPAnt-2, a peptide antagonistic to Gs proteins that stimulates adenylate cyclase. Neither of these latter inhibitors, however, affected JH III-evoked patency. These results suggest that, in the process of patency in H. virescens ovarial follicles, JH I predominantly signals via the cAMP-dependent second messenger system, whereas JH III acts via the inositol triphosphate/diacylglycerol signaling pathway. Moreover, stimulation of patency by cholera toxin alone and inhibition of JH I-evoked patency by GPAnt-2, strongly suggest that JH I acts on the follicular epithelial cells via activation of G-protein, and-possibly-via G(s)-protein coupled receptor.