A 2.1 A resolution structure of an uncleaved alpha(1)-antitrypsin shows variability of the reactive center and other loops

Serpin (serine protease inhibitor) proteins are involved in diverse physiological processes including inflammation, coagulation, matrix remodeling, and cell differentiation. Deficiency of normal serpin functions leads to various hereditary diseases. Besides their clinical importance, serpin proteins draw much attention due to the large conformational changes that occur upon interaction with proteases. We present here the crystal structure of an uncleaved alpha(1)-antitrypsin determined by the multiple isomorphous replacement method and refined to 2.1 A resolution. The structure, which is the first active serpin structure based on experimental phases, reveals novel conformations in the flexible loops, including the proximal hinge region of the reactive center loop and the surface cavity region in the central beta-sheet, sheet A. The determined loop conformation explains the results of recent mutagenesis studies and provides detailed insights into the protease inhibition mechanism. The high-resolution structure of active alpha(1)-antitrypsin also provides evidence for the existence of localized van-der-Waals strain in the central hydrophobic core.     

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.     

EVIDENCE FOR SEXUAL REPRODUCTION IN THE MARINE DINOFLAGELLATES, PYROCYSTIS NOCTILUCA AND PYROCYSTIS LUNULA (DINOPHYTA)

Evidence for sexual reproduction was observed in two oceanic dinoflagellate species, Pyrocystis noctiluca Murray ex Haeckel and Pyrocystis lunula (Schütt) Schütt. Observations suggest that cells underwent fertilization as opposed to cell division because of the following: first, fusing cells had a conspicuous pore (fusion pore) connecting the two gametes; dividing cells lacked this feature. In culture, about 0.1% of P. noctiluca cells had a fusion pore, which serves as a possible site for gamete recognition on the cell wall. Second, we document a temporal progression of plasmogamy and karyogamy. Fusion events in both species were observed at the beginning of the day, whereas division stages were most apparent at the end of the day.     

4-1BB-mediated immunotherapy of rheumatoid arthritis

Collagen type II-induced arthritis is a CD4(+) T-cell-dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4(+) T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c(+)CD8(+) T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b(+) monocytes and CD11c(+) dendritic cells. Both anti-interferon-gamma and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c(+)CD8(+) T cells, and that interferon-gamma produced by these cells suppresses antigen-specific CD4(+) T cells through an indoleamine 2,3-dioxygenase-dependent mechanism.     

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni²⁺-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP₂) and its activity was Ca²⁺-dependent, which was similar to mammalian PLCδ isozymes.     

Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin

Expression with a fusion partner is now a popular scheme to produce a protein of interest because it provides a generic tool for expression and purification. In our previous study, a strong polycationic tail has been harnessed for an efficient purification scheme. Here, the same polycation tail attached to a protein of interest is shown to hold versatility for a solid-phase refolding method that utilizes a charged adsorbent as a supporting material. Cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at the C-terminus (CGTK10ase) retains the ability to bind to a cation exchanger even in a urea-denatured state. When the denatured and adsorbed CGTK10ase is induced to refold, the bound CGTK10ase aggregates little even at a g/L range. The renatured CGTK10ase can also be simply recovered from the solid support by adding high concentration of NaCl. The CGTK10ase refolded on a solid support retains specific enzyme activity virtually identical to that of the native CGTK10ase. Several factors that are important in improving the refolding efficiency are explored. Experimental results indicate that nonspecific electrostatic interactions between the charge of the ion exchanger and the local charge of CGTase other than the polycationic tag should be reduced to obtain higher refolding yield. The solid-phase refolding method utilizing a strong polycationic tag resulted in a remarkable increase in the refolding performance. Taken together with the previous report in which a series of polycations were explored for efficient purification, expression of a target protein fused with a strong polycation provides a straightforward protein preparation scheme.     

Pegasys: software for executing and integrating analyses of biological sequences

Background  

We present Pegasys – a flexible, modular and customizable software system that facilitates the execution and data integration from heterogeneous biological sequence analysis tools.