Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum.

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.

Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter.

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.

Expression and activity of the nucleotide-binding domains of the human ABCA1 transporter.

The aim of this study was the expression and production in Escherichia coli of the nucleotide-binding domains (NBDs) of the human ABCA1 transporter, in a soluble, non-denatured form. To increase the protein solubility, and avoid expression in E. coli inclusion bodies, we extended the length of the expressed NBD domains, to include proximal domains. The corresponding cDNA constructs were used to express the N-terminal His-tagged WT and mutant proteins, which were purified by Ni(2+)-affinity chromatography. Optimal expression of soluble proteins was obtained for constructs including the NBD, the downstream 80-residue domain, and about 20 upstream residues. The size homogeneity of WT and mutant NBDs was determined by Dynamic Light Scattering, and ATP-binding constants and ATPase activities were measured. The NBD1 and NBD2 domains bound ATP with comparable affinity. The ATPase activity of WT His-NBD1 was about three times higher than that of NBD2 and amounted to 5913 compared to 1979 nmol Pi/micromol NBD/min for WT His-NBD2. All engineered mutants had comparable ATPase activity to the corresponding WT protein. The optimisation of the length of the expressed proteins, based upon the boundary prediction of NBDs and neighbour domains, enables the expression and purification of soluble ABCA1 NBDs, with high ATPase activity. This approach should prove useful for the study of the structural and functional properties of the NBDs and other domains of the ABC transporters.

Characterization of the ABCA transporter subfamily: identification of prokaryotic and eukaryotic members, phylogeny and topology.

An alignment of the mammalian ABCA transporters enabled the identification of sequence segments, specific to the ABCA subfamily, which were used as queries to search for eukaryotic and prokaryotic homologues. Thirty-seven eukaryotic half and full-length transporters were found, and a close relationship with prokaryotic subfamily 7 transporters was detected. Each half of the ABCA full-transporters is predicted to comprise a membrane-spanning domain (MSD) composed of six helices and a large extracellular loop, followed by a nucleotide-binding domain (NBD) and a conserved cytoplasmic 80-residue sequence, which might have a regulatory function. The topology predicted for the ABCA transporters was compared to the crystal structures of the MsbA and BtuCD bacterial transporters. The alignment of the MSD and NBD domains provided an estimate of the degree of residue conservation in the cytoplasmic, extracellular and transmembrane domains of the ABCA transporter subfamily. The phylogenic tree of eukaryotic ABCA transporters based upon the NBD sequences, consists of three major clades, corresponding to the half-transporter single NBDs and to the full-transporter NBDls and NBD2s. A phylogenic tree of prokaryotic transporters and the eukaryotic ABCA transporters confirmed the evolutionary relationship between prokaryotic subfamily 7 transporters and eukaryotic ABCA half and full-transporters.

In vitro studies of Flemish, Dutch, and wild-type beta-amyloid provide evidence for two-staged neurotoxicity.

Mutations in the beta-amyloid (Abeta) sequence of the amyloid precursor protein gene (APP) present with variable disease phenotypes. While patients with the Dutch APP mutation (E693Q) have predominantly hemorrhagic strokes, Flemish APP (A692G) patients develop both strokes and Alzheimer's disease (AD). To determine whether these diverse clinical and pathological presentations are due to mutant Abeta or APP, we studied the effect of Flemish, Dutch, and wild-type Abeta/APP on phosphorylation of specific tau epitopes observed in AD. No effect was observed in differentiated SH-SY5Y cells either stably expressing APP or treated with synthetic Abeta(12-42). However, we did observe a paradoxical temporal difference in the neurotoxic potential of mutant and wild-type Abeta. While long 24-h incubation at physiological levels of Abeta (2 microM) showed a higher amount of apoptosis for Dutch Abeta, a short 2-h incubation showed elevated apoptosis for Flemish and wild-type Abeta. The altered aggregating properties of Abeta, with Dutch Abeta aggregating faster and Flemish Abeta slower than wild type, elucidated a discrete two-phase Abeta neurotoxicity. We propose here that, at least in vitro, Abeta might be neurotoxic in an initial phase due to its soluble oligomeric or other early toxic Abeta intermediate(s), which is perhaps distinct from the late neurotoxicity incurred by aggregated larger assemblies of Abeta.