Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers

BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t((1/2))) of 11-14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.     

Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines

Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.     

ruvA and ruvB mutants specifically impaired for replication fork reversal

Replication fork reversal (RFR) is a reaction that takes place in Escherichia coli at replication forks arrested by the inactivation of a replication protein. Fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a Holliday junction adjacent to DNA double-strand end, both of which are processed by recombination enzymes. In several replication mutants, replication fork reversal is catalysed by the RuvAB complex, originally characterized for its role in the last steps of homologous recombination, branch migration and resolution of Holliday junctions. We present here the isolation and characterization of ruvA and ruvB single mutants that are impaired for RFR at forks arrested by the inactivation of polymerase III, while they remain capable of homologous recombination. The positions of the mutations in the proteins and the genetic properties of the mutants suggest that the mutations affect DNA binding, RuvA-RuvB interaction and/or RuvB-helicase activity. These results show that a partial RuvA or RuvB defect affects primarily RFR, implying that RFR is a more demanding reaction than Holliday junction resolution.     

Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.     

Cytogenetic evolution of human ovarian cell lines associated with chemoresistance and loss of tumorigenicity.

In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH) to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex-FISH and Spectral Karyotype) and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug-resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8)t(8;13)(p22;q?) and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8)) or the loss of tumorigenicity properties (del(11p)).     

The unique headpore of Monospilus dispar (Cladocera,Chydoridae) is not lonesome: new record of minute lateral pores

The headshield of Monospilus dispar (Cladocera, Chydoridae, Aloninae) was characterized by the presence of a unique headpore. Scanning Electron Microscopy shows the occurrence of minute pores close to the main headpore. The intimate structure of the main headpore is formed by concentric lamellae. This new information leads to new considerations about phylogeny.     

Evidence that the conformational stability of 'aged' organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called 'aged' enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60 degrees C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20 degrees C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0 degree C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0 degree C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.