Crystal structure of the hypothetical protein TA1238 from Thermoplasma acidophilum: a new type of helical super-bundle

The crystal structure of the hypothetical protein TA1238 from Thermoplasma acidophilum was solved with multiple-wavelength anomalous diffraction and refined at 2.0 A resolution. The molecule consists of a typical four-helix antiparallel bundle with overhand connection. However, its oligomerization into a trimer leads to a coiled "super-helix" which is novel for such bundles. Its central feature, a six-stranded coiled coil, is also novel for proteins. TA1238 does not have strong sequence homologues in databases, but shows strong structural similarity with some proteins in the Protein Data Bank. The function could not be inferred from the sequence but the structure, with some rearrangement, bears some resemblance to the active site region of cobalamin adenosyltransferase (TA1434). Specifically, TA1238 retains Arg104, which is structurally equivalent to functionally critical Arg119 of TA1434. For such conformational change, the overhand connection of TA1238 might need to be involved in a gating mechanism that might be modulated by ligands and/or by interactions with the physiological partners. This allowed us to hypothesize that TA1238 could be involved in cobalamin biosyntheses.     

Mitochondrial proteins and complexes in Leishmania and Trypanosoma involved in U-insertion/deletion RNA editing

A number of mitochondrial proteins have been identified in Leishmania sp. and Trypanosoma brucei that may be involved in U-insertion/deletion RNA editing. Only a few of these have yet been characterized sufficiently to be able to assign functional names for the proteins in both species, and most have been denoted by a variety of species-specific and laboratory-specific operational names, leading to a terminology confusion both within and outside of this field. In this review, we summarize the present status of our knowledge of the orthologous and unique putative editing proteins in both species and the functional motifs identified by sequence analysis and by experimentation. An online Supplemental sequence database (http://164.67.60.200/proteins/protsmini1.asp) is also provided as a research resource.     

COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance

We present a novel method for the comparison of multiple protein alignments with assessment of statistical significance (COMPASS). The method derives numerical profiles from alignments, constructs optimal local profile-profile alignments and analytically estimates E-values for the detected similarities. The scoring system and E-value calculation are based on a generalization of the PSI-BLAST approach to profile-sequence comparison, which is adapted for the profile-profile case. Tested along with existing methods for profile-sequence (PSI-BLAST) and profile-profile (prof_sim) comparison, COMPASS shows increased abilities for sensitive and selective detection of remote sequence similarities, as well as improved quality of local alignments. The method allows prediction of relationships between protein families in the PFAM database beyond the range of conventional methods. Two predicted relations with high significance are similarities between various Rossmann-type folds and between various helix-turn-helix-containing families. The potential value of COMPASS for structure/function predictions is illustrated by the detection of an intricate homology between the DNA-binding domain of the CTF/NFI family and the MH1 domain of the Smad family.     

A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.     

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.     

Human copper-transporting ATPase ATP7B (the Wilson's disease protein): biochemical properties and regulation

Wilson's disease protein (WNDP) is a product of a gene ATP7B that is mutated in patients with Wilson's disease, a severe genetic disorder with hepatic and neurological manifestations caused by accumulation of copper in the liver and brain. In a cell, WNDP transports copper across various cell membranes using energy of ATP-hydrolysis. Copper regulates WNDP at several levels, modulating its catalytic activity, posttranslational modification, and intracellular localization. This review summarizes recent studies on enzymatic function and copper-dependent regulation of WNDP. Specifically, we describe the molecular architecture and major biochemical properties of WNDP, discuss advantages of the recently developed functional expression of WNDP in insect cells, and summarize the results of the ligand-binding studies and molecular modeling experiments for the ATP-binding domain of WNDP. In addition, we speculate on how copper binding may regulate the activity and intracellular distribution of WNDP, and what role the human copper chaperone Atox1 may play in these processes.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.     

Speciation and Bioavailability Measurements of Environmental Plutonium Using Diffusion in Thin Films

The biological uptake of plutonium (Pu) in aquatic ecosystems is of particular concern since it is an alpha-particle emitter with long half-life which can potentially contribute to the exposure of biota and humans. The diffusive gradients in thin films technique is introduced here for in-situ measurements of Pu bioavailability and speciation. A diffusion cell constructed for laboratory experiments with Pu and the newly developed protocol make it possible to simulate the environmental behavior of Pu in model solutions of various chemical compositions. Adjustment of the oxidation states to Pu(IV) and Pu(V) described in this protocol is essential in order to investigate the complex redox chemistry of plutonium in the environment. The calibration of this technique and the results obtained in the laboratory experiments enable to develop a specific DGT device for in-situ Pu measurements in freshwaters. Accelerator-based mass-spectrometry measurements of Pu accumulated by DGTs in a karst spring allowed determining the bioavailability of Pu in a mineral freshwater environment. Application of this protocol for Pu measurements using DGT devices has a large potential to improve our understanding of the speciation and the biological transfer of Pu in aquatic ecosystems.