Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties

Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.     

Prolonged prenatal hypernatremia alters neuroendocrine and electrolyte homeostasis in neonatal sheep

Arginine vasopressin (AVP) is a neuroendocrine hormone synthesized in the hypothalamus, and is stored and secreted by the posterior pituitary gland in response to stimuli such as plasma hypertonicity and hypotension. The primary physiologic roles of AVP include plasma osmolality and blood pressure regulation. We have previously demonstrated that chronic prenatal plasma hypertonicity alters the AVP regulatory pathway in newborn lambs. The objectives of the present study were to evaluate prolonged effects of antenatal plasma hypertonicity on neonatal plasma osmoregulation. Pregnant ewes at 119 +/- 3 days of gestation were water restricted to achieve and maintain hypertonicity until normal-term delivery. After delivery, ewes were provided food and water ad libitum and lambs were allowed maternal nursing. At the age of 28 days, blood samples were obtained for the analysis of plasma osmolality, electrolytes, and AVP levels from study (n= 5) and age-matched control (n= 6) lambs. Subsequently, lambs were euthanized, and the pituitary and hypothalamus were processed for the determination of pituitary AVP content by radioimmunoassay, and AVP gene expression by Northern analysis. In response to water restriction, maternal plasma osmolality significantly increased (306 +/- 1.1 to 326 +/- 1.2 mOsm/kg, P< 0.001). At the age of 28 days, plasma sodium level was higher in study (prenatally dehydrated) than control lambs (144.6 +/- 0.4 vs 142.6 +/- 0.3,P< 0.05). Study lambs had higher plasma AVP concentrations than the control lambs (4.1 +/- 0.4 vs 1.7 +/- 0.4 pg/ml,P< 0.05). Similarly, total pituitary AVP content was higher in thein utero hypertonic lambs than in the control lambs (6.5 +/- 1.0 vs 2.8 +/-1.2 microg, P< 0.05). However, there was no difference in hypothalamic AVP mRNA levels between the two groups. The present study demonstrates that chronic maternal and fetal plasma hypertonicity has prolonged effects on pituitary and plasma AVP, as well as plasma sodium in neonatal lambs, providing further evidence suggesting prenatal imprinting of osmoregulation through at least 1 month of age.     

Deletion of secretory group V phospholipase A2 attenuates cell migration and airway hyperresponsiveness in immunosensitized mice

We investigated the role of group V phospholipase A2 (gVPLA2) in OVA-induced inflammatory cell migration and airway hyperresponsiveness (AHR) in C57BL/6 mice. Repeated allergen challenge induced biosynthesis of gVPLA2 in airways. By aerosol, gVPLA2 caused dose-related increase in airway resistance in saline-treated mice; in allergic mice, gVPLA2 caused persistent airway narrowing. Neither group IIa phospholipase A2, a close homolog of gVPLA2, nor W31A, an inactive gVPLA2 mutant with reduced activity, caused airway narrowing in immune-sensitized mice. Pretreatment with MCL-3G1, a blocking Ab against gVPLA2, before OVA challenge blocked fully gVPLA2-induced cell migration and airway narrowing as marked by reduction of migrating leukocytes in bronchoalveolar lavage fluid and decreased airway resistance. We also assessed whether nonspecific AHR caused by methacholine challenge was elicited by gVPLA2 secreted from resident airway cells of immune-sensitized mice. MCL-3G1 also blocked methacholine-induced airway bronchoconstriction in allergic mice. Blockade of bronchoconstriction by MCL-3G1 was replicated in allergic pla2g5-/- mice, which lack the gene encoding gVPLA2. Bronchoconstriction caused by gVPLA2 in pla2g4-/- mice was comparable to that in pla2g4+/+ mice. Our data demonstrate that gVPLA2 is a critical messenger enzyme in the development of AHR and regulation of cell migration during immunosensitization by a pathway that is independent of group IVa phospholipase A2.     

Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.     

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies

MamMiBase, the mammalian mitochondrial genome database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phylogenetic analysis, since it allows a ready retrieval of nucleotide and aminoacid individual alignments, in three different formats (NEXUS for PAUP program, for MEGA program and for PHYLIP program) of the 13 protein coding mitochondrial genes. The user may download the sequences that are useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, which are also given by MamMiBase. A simple phylogenetic tree (neighbor-joining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. AVAILABILITY: MamMiBase is available at http://www.mammibase.lncc.br     

Human mast cell-derived gelatinase B (matrix metalloproteinase-9) is regulated by inflammatory cytokines: role in cell migration

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid arthritis (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and proteases that are released upon activation. This study investigated the regulation of matrix metalloproteinase MMP-9 (gelatinase B) in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-alpha significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-gamma inhibited MMP-9 mRNA and protein expression. The cytokine-mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse bone marrow-derived mast cells. Furthermore, TNF-alpha significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-gamma, rTIMP-1, or pharmacological MMP inhibitors significantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this enzyme under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage.     

Structural evidence for a proton transfer pathway coupled with haem reduction of cytochrome c″ from Methylophilus methylotrophus

The crystal structures of the oxidized and reduced forms of cytochrome c″ from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation in the much more complex protein cytochrome c oxidase.     

Group V secretory phospholipase A2 amplifies the induction of cyclooxygenase 2 and delayed prostaglandin D2 generation in mouse bone marrow culture-derived mast cells in a strain-dependent manner

Activation of mouse bone marrow-derived mast cells (BMMC) with stem cell factor (SCF) or IgE and antigen elicits exocytosis and an immediate phase of prostaglandin (PG) D(2) and leukotriene (LT) C(4) generation. Activation of BMMC by SCF, IL-1beta and IL-10 elicits a delayed phase of PGD(2) generation dependent on cyclooxygenase (COX) 2 induction. Cytosolic phospholipase A(2) alpha provides arachidonic acid in both phases and amplifies COX-2 induction. Pharmacological experiments implicate an amplifying role for secretory (s) PLA(2). We used mice lacking the gene encoding group V sPLA(2) (Pla2g5-/-) to definitively test its role in eicosanoid generation by BMMC. Pla2g5-/- BMMC on a C57BL/6 genetic background showed a modest reduction in exocytosis and immediate PGD(2) generation after activation with SCF or with IgE and antigen, while LTC(4) generation was not modified. Delayed-phase PGD(2) generation and COX-2 induction were reduced approximately 35% in C57BL/6 Pla2g5-/- BMMC and were restored by exogenous PGE(2). There was no deficit in either phase of eicosanoid generation by Pla2g5-/- BMMC on a BALB/c background. Thus, group V sPLA(2) amplifies COX-2 expression and delayed phase PGD(2) generation in a strain-dependent manner; it has at best a limited role in immediate eicosanoid generation by BMMC.