Molecular control of phenoloxidase-induced melanin synthesis in an insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Sp?tzle-processing enzyme is a key enzyme in the production of cleaved Sp?tzle from pro-Sp?tzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Sp?tzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.     

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.     

Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.     

A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna

A small conserved open reading frame in the plastid genome, ycf9, encodes a putative membrane protein of 62 amino acids. To determine the function of this reading frame we have constructed a knockout allele for targeted disruption of ycf9. This allele was introduced into the tobacco plastid genome by biolistic transformation to replace the wild-type ycf9 allele. Homoplasmic ycf9 knockout plants displayed no phenotype under normal growth conditions. However, under low light conditions, their growth rate was significantly reduced as compared with the wild-type, due to a lowered efficiency of the light reaction of photosynthesis. We show that this phenotype is caused by the deficiency in a pigment-protein complex of the light-harvesting antenna of photosystem II and hence by a reduced efficiency of photon capture when light availability is limiting. Our results indicate that, in contrast to the current view, light-harvesting complexes do not only consist of the classical pigment-binding proteins, but may contain small structural subunits in addition. These subunits appear to be crucial architectural factors for the assembly and/or maintenance of stable light-harvesting complexes.     

A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of orf1 was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.     

A 43-kDa protein of Treponema denticola is essential for dentilisin activity

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.     

Crystal structure of a novel regulatory 40-kDa mammary gland protein (MGP-40) secreted during involution

We have determined the crystal structure of a novel regulatory protein (MGP-40) from the mammary gland. This protein is implicated as a protective signaling factor that determines which cells are to survive the drastic tissue remodeling that occurs during involution. It has been indicated that certain cancers could surreptitiously utilize the proposed normal protective signaling by proteins of this family to extend their own survival and thereby allow them to invade the organ and metastasize. In view of this, MGP-40 could form an important target for rational structure-based drug design against breast cancer. It is a single chain, glycosylated protein with a molecular mass of 40 kDa. It was isolated from goat dry secretions and has been cloned and sequenced. It was crystallized by microdialysis from 20 mg ml(-1) solution in 0.1 m Tris-HCl, pH 8.0, and equilibrated against the same solution containing 19% ethanol. Its x-ray structure has been determined by molecular replacement and refined to a 2.9 A resolution. The protein adopts a beta/alpha domain structure with a triose-phosphate isomerase barrel conformation in the core and a small alpha+beta folding domain. A single glycosylation site containing two N-acetylglucosamine units has been observed in the structure. Compared with chitinases and chitinase-like proteins the most important mutation in this protein pertains to a change from Glu to Leu at position 119, which is part of the so-called active site sequence in the form of Asp(115), Leu(119), and Asp(186) and in this case resulting in the loss of chitinase activity. The orientations of two Trp residues Trp(78) and Trp(331) in the beta barrel reduces the free space, drastically impairing the binding of saccharides/polysaccharides. However, the site and mode of binding of this protein to cell surface receptors are not yet known.     

A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide

Determination of structures and functions of pattern recognition proteins are important for understanding pathogen recognition mechanisms in host defense and for elucidating the activation mechanism of innate immune reactions. In this study, a novel 40-kDa protein, named LPS recognition protein (LRP), was purified to homogeneity from the cell-free plasma of larvae of the large beetle, Holotrichia diomphalia. LRP exhibited agglutinating activities on Escherichia coli, but not on Staphylococcus aureus and Candida albicans. This E. coli-agglutinating activity was preferentially inhibited by the rough-type LPS with a complete core oligosaccharide. LRP consists of 317 aa residues and six repeats of an epidermal growth factor-like domain. Recombinant LRP expressed in a baculovirus system also showed E. coli agglutination activity in vitro and was able to neutralize LPS by inhibition of LPS-induced IL-6 production in mouse bone marrow mast cells. Furthermore, E. coli coated with the purified LRP were more rapidly cleared in the Holotrichia larvae than only E. coli, indicating that this protein participates in the clearance of E. coli in vivo. The three amino-terminal epidermal growth factor-like domains of LRP, but not the three carboxyl epidermal growth factor-like domains, are involved in the LPS-binding activity. Taken together, this LRP functions as a pattern recognition protein for LPS and plays a role as an innate immune protein.     

Design and synthesis of substituted quinolines as novel and selective melanin concentrating hormone antagonists as anti-obesity agents

A novel series of substituted quinoline analogs were designed and synthesized as potent and selective melanin concentrating hormone (MCH) antagonists. These analogs show potent (nM) activity (12a-k) with a moderate selectivity. Conversely, the conformationally constrained thienopyrimidinone analogs (18a-g) showed improved activity in MCH-1R and selectivity over 5HT2C.     

Lamprey 48-kDa lens protein represents a novel class of crystallins

SDS-PAGE revealed a major Mr 48 000 polypeptide of pI around 8 in the water-soluble fraction of lamprey lenses. It occurs as a monomeric protein, and its amino acid composition and tryptic peptides show no resemblances to alpha-, beta-, gamma- or delta-crystallin. Immunoblotting with antiserum against the 48-kDa protein revealed an immunologically related polypeptide of similar Mr in reptiles, several birds and a fish, but showed no cross-reactivity with any other water-soluble lens component. The 48-kDa protein is not detected in many birds and fishes, and in the investigated mammals and amphibians.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

Specificity of nuclear protein binding to a CYP1A1 negative regulatory element

Primary cultures of rat epidermal keratinocytes lose the ability to respond to chemicals with the induction of CYP1A1 gene expression after approximately 15 passages. This repression is mediated by a CT-rich direct repeat negative regulatory DNA (NeRD) element present in the upstream regulatory region of the CYP1A1 gene. Competitive gel retardation analysis using keratinocyte nuclear extracts and mutant NeRD oligonucleotides revealed the presence of two specific protein-NeRD complexes and revealed the specific nucleotides important for the formation of each complex. These studies demonstrate that these two factors bind to overlapping sites within the NeRD element. Nucleotide specificity of complex A formation is similar to that of previously identified nuclear silencing factors, while that of complex B appears to represent a unique CT-rich binding factor. These results suggest that repression of CYP1A1 gene expression in high passage keratinocytes may involve the interplay between at least two specific NeRD binding factors.     

The chloroplast reductase-binding protein is identical to the 16.5-kDa polypeptide described as a component of the oxygen-evolving complex

The N-terminal sequence of the spinach chloroplast reductase-binding protein was determined. The sequence is the same one of a 16.5-kDa polypeptide described as a component of the oxygen-evolving system. Antibodies against both proteins are equivalent as shown by immunoblots, Ouchterlony assays, precipitation of reductase-binding protein complex, and agglutination of thylakoids partially depleted of reductase. These results suggest both proteins are identical. Exposure of the binding protein on the stromal side of thylakoids is supported by agglutination of thylakoids partially depleted of reductase, proteolysis by trypsin, and by accessibility to Fab of anti-binding protein. The latter prevents rebinding of reductase supporting the functional role of the binding protein (16.5 kDa).     

A novel cell-free protein synthesis system

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30–45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.