Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function.

Reactive oxygen and nitrogen intermediates can cause damage to many cellular components and have been implicated in a number of diseases. Cells have developed a variety of mechanisms to destroy these reactive molecules or repair the damage once it occurs. In proteins one of the amino acids most easily oxidized is methionine, which is converted to methionine sulfoxide. An enzyme, peptide methionine sulfoxide reductase (MsrA), catalyzes the reduction of methionine sulfoxide in proteins back to methionine. There is growing evidence that MsrA plays an important role in protecting cells against oxidative damage. This paper reviews the biochemical properties and biological role of MsrA.     

Common Mode of Remodeling AAA ATPases p97/CDC48 by Their Disassembling Cofactors ASPL/PUX1

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Staining Chromosomes in Sections of Germinal Vesicles of Sarcoptid Mites by a Schiff Reagent

Chromosomes of oocytes, especially early prophase I stages, of Acaridae and Anoetidae species are difficult to stain by procedures using hematoxylin, Feulgen and aceto-orcein. Hematoxylin stains are intensely polychromatic in oocytes; the standard Feulgen procedure is negative with chromosomes during diffuse prophase stages. Satisfactory staining can be obtained with a supersensitive Schiff reagent (Tobie, W. C., Ind. Eng. Chem., Anal. Ed., 14: 405—406, 1942) made by reducing basic fuchsin with gaseous SO₂. Routinely prepared paraffin sections of mites fixed in Carnoy's 6:3:1 mixture were hydrolysed 5-8 min in 1 N HCl, washed well, and stained in this reagent: 1-2 hr for prophase oocytes, 10-20 min for condensed chromosomes. A second staining in a 0.5% aqueous solution of toluidine blue 0, adjusted to pH 5.3-5.5 with a citrate buffer, served to darken the original Feulgen stain. Counterstaining with 0.1-0.2% fast green FCF in the last fluid of the dehydrating series enhanced contrast between chromosomes and cytoplasm. This staining technic is also suitable for preparing whole mounts of mites.     

Two-pore channels form homo- and heterodimers

Two-pore channels (TPCs) have been recently identified as NAADP-regulated Ca(2+) release channels, which are localized on the endolysosomal system. TPCs have a 12-transmembrane domain (TMD) structure and are evolutionary intermediates between the 24-TMD α-subunits of Na(+) or Ca(2+) channels and the transient receptor potential channel superfamily, which have six TMDs in a single subunit and form tetramers with 24 TMDs as active channels. Based on this relationship, it is predicted that TPCs dimerize to form functional channels, but the dimerization of human TPCs has so far not been studied. Using co-immunoprecipitation studies and a mass spectroscopic analysis of the immunocomplex, we show the presence of homo- and heteromeric complexes for human TPC1 and TPC2. Despite their largely distinct localization, we identified a discrete number of endosomes that coexpressed TPC1 and TPC2. Homo- and heteromerization were confirmed by a FRET study, showing that both proteins interacted in a rotational (N- to C-terminal/head-to-tail) symmetry. This is the first report describing the presence of homomultimeric TPC1 channels and the first study showing that TPCs are capable of forming heteromers.     

Structure in solution of a four-helix lipid binding protein.

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H-1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi 1 angles, and 2 chi 2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A +/- 0.04 A for backbone atoms and 0.93 A +/- 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Structure 3:189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.     

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.     

Estimation of Na+ dwell time in the gramicidin A channel. Na+ ions as blockers of H+ currents

The permeation of Na+ through gramicidin A channels shows a simple saturation with increasing Na+ concentration that can be described by two different models. The first model assumes that one Na+ binds to the channel with high affinity (approximately 30 M-1) and that conduction occurs by a 'knock-on' mechanism requiring double occupancy of the channel; the other model assumes that Na+ binding is of low affinity (less than 1 M-1), and that double occupancy of the channel is rare. NMR measurements have shown tight Na+ binding, favoring the first model, but measurements of flux ratios and water transport support the second model. We present here a relatively model-independent measurement of the dwell time of Na+ inside the channel, in which we characterize the fluctuations in H+ current through the channel induced by 'block' from the more slowly permeating Na+ ions. The mean Na+ dwell time inside the channel is estimated to be approximately 10 ns at a membrane potential of 200 mV. This result is inconsistent with tight Na+ binding, thus favoring the second model.     

Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1^fd, which is cleaved off after mitochondrial import. The physiological function of Etp1^fd is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1^fd has a melting temperature more than 20 °C higher than adrenodoxin. The three-dimensional structure of Etp1^fd has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1^fd with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1^fd and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.