A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alphabeta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alphabeta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alphabeta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alphabeta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting 'competitive' antagonist) binds to this residue.     

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Integration of the Mitochondrial-Processing Peptidase into the Cytochrome bc 1 Complex in Plants

The plant mitochondrial cytochrome bc ₁ complex, like nonplant mitochondrial complexes,consists of cytochromes b and c ₁, the Rieske iron–sulfur protein, two Core proteins, and fivelow-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteinsare identical to subunits of the general mitochondrial processing peptidase (MPP). The MPPis a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptidesfrom hundreds of the nuclear-encoded mitochondrial precursor proteins that are synthesizedin the cytosol and imported into the mitochondrion. Integration of the MPP into the bc ₁complex renders the bc ₁ complex in plants bifunctional, being involved both in electrontransport and in protein processing. Despite the integration of MPP into the bc ₁ complex,electron transfer as well as translocation of the precursor through the import channel areindependent of the protein-processing activity. Recognition of the processing site by MPPoccurs via the recognition of higher-order structural elements in combination with charge andcleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammaliancytochrome bc ₁ complex is highly useful for understanding of the mechanism of action of MPP.In memory of my teacher—an insightful, devoted, and enthusiastic scientist and an amiable and kind-hearted human being—Lars Ernster     

Structural Basis of Multifunctional Bovine Mitochondrial Cytochrome bc 1 Complex

The mitochondrial cytochrome bc ₁ complex is a multifunctional membrane protein complex. Itcatalyzes electron transfer, proton translocation, peptide processing, and superoxide generation.Crystal structure data at 2.9 Å resolution not only establishes the location of the redox centersand inhibitor binding sites, but also suggests a movement of the head domain of the iron–sulfurprotein (ISP) during bc ₁ catalysis and inhibition of peptide-processing activity during complexmaturation. The functional importance of the movement of extramembrane (head) domain ofISP in the bc ₁ complex is confirmed by analysis of the Rhodobacter sphaeroides bc ₁ complexmutants with increased rigidity in the ISP neck and by the determination of rate constants foracid/base-induced intramolecular electron transfer between [2Fe–2S] and heme c ₁ in nativeand inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovineheart mitochondrial bc ₁ complex by nonionic detergent at concentrations that inactivate electrontransfer activity. This peptide-processing activity is shown to be associated with subunits Iand II by cloning, overexpression and in vitro reconstitution. The superoxide-generation siteof the cytochrome bc ₁ complex is located at reduced b _L and Q^–. The reaction is membranepotential-, and cytochrome c-dependent.     

Molecular mechanism of voltage sensor movements in a potassium channel

Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization--this motion is accompanied by a reorientation of S4 charges to the extracellular phase.     

Letter to the Editor: 1H, 15N and 13C NMR Resonance Assignments of Staphostatin A, a Specific Staphylococcus Aureus Cysteine Proteinase Inhibitor

The increasing antibiotic resistance of an important human pathogen Staphylococcus aureus calls for the development of new therapeutic strategies. Staphylococcal cysteine proteases have been suggested as targets for such therapies. The recent discovery of staphostatins, specific protein inhibitors of these enzymes, gives prospects for the design and production of synthetic, low molecular weight analogs which might become drugs. We have decided to structurally characterize staphostatin A, a representative inhibitor of staphylococcal cysteine proteases, and to assess its binding mode to the target protease with the view of clarifying the specificity determinants. Here we report the (1)H, (15)N and (13)C NMR resonance assignments of staphostatin A.     

Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay

A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.     

Isolation and Characterization of a Rice Cysteine Protease Gene, OsCP1, Using T-DNA Gene-Trap System

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.