Enzymatic activity of the SARS coronavirus main proteinase dimer

The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH)(2)-Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K(m), and is unusually sensitive to ionic strength and reducing agents.     

An outer membrane enzyme encoded by Salmonella typhimurium lpxR that removes the 3'-acyloxyacyl moiety of lipid A

The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3'-acyloxyacyl residue of lipid A in the presence of 5 mM Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3'-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3'-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3'-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3'-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli O157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.     

Four and a half LIM protein 1 binds myosin-binding protein C and regulates myosin filament formation and sarcomere assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced "sac-like" myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.     

Structure and biosynthesis of free lipid A molecules that replace lipopolysaccharide in Francisella tularensis subsp. novicida

Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an alpha-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An alpha-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.     

PERK EIF2AK3 control of pancreatic beta cell differentiation and proliferation is required for postnatal glucose homeostasis

Mutations in PERK (EIF2AK3) result in permanent neonatal diabetes as well as several other anomalies that underlie the human Wolcott-Rallison syndrome, and these anomalies are mirrored in Perk knockout mice. To identify the cause of diabetes in PERK-deficient mice, we generated a series of tissue- and cell-specific knockouts of the Perk gene and performed a developmental analysis of the progression to overt diabetes. We discovered that PERK is specifically required in the insulin-secreting β cells during the fetal and early neonatal period as a prerequisite for postnatal glucose homeostasis. However, PERK expression in β cells is not required at the adult stage to maintain β cell functions and glucose homeostasis. We show that PERK-deficient mice exhibit severe defects in fetal/neonatal β cell proliferation and differentiation, resulting in low β cell mass, defects in proinsulin trafficking, and abrogation of insulin secretion that culminate in permanent neonatal diabetes.     

Foliar pubescence and resistance to potato moth, Phthorimaea operculella, in Lycopersicon hirsutum

Pubescence characteristics for six accessions of Lycopersicon hirsutum Dunal and five accessions of L. hirsutum f. glabratum CH Mull. were determined and compared with those of an accession of cultivated tomato (L. esculentum Mill.). Removal of trichome exudates from excised leaflets using ethanol solution resulted in a reduced mortality and increased survival of potato moth (Phthorimaea operculella (Zeller)) neonates for the accessions that were most lethal when not treated with ethanol solution. No such treatment effect was evident for L. esculentum or for the L. hirsutum accession with least effect on neonates when its trichomes were intact. In a glasshouse experiment with caged intact plants, mortality of neonate P. operculella placed on the abaxial surface was greater on seven accessions than for L. esculentum.Neonates were less severely affected on the adaxial surface. Eleven days after inoculation, no live larvae were found on LA 1927, PI 127827, PI 134418, and PI 134428, and numbers on other accessions were lower than for L. esculentum. Eventual emergence of adults followed a similar trend. Multiple regression of insect data against pubescence indicated a significant correlation between density of type IV and VI trichomes and neonate mortality, decreased larval development and decreased adult emergence. Non-glandular type V trichomes were positively correlated with high survival of insects to 11 days and to adult. Though factors other than glandular trichomes are likely to be important, increased density of type IV and VI, along with reduced type V, are shown to be important to select in breeding for P. operculella resistance.