James Féthière

Bacterial polysaccharide co-polymerases share a common framework for control of polymer length

The chain length distribution of complex polysaccharides present on the bacterial surface is determined by polysaccharide co-polymerases (PCPs) anchored in the inner membrane. We report crystal structures of the periplasmic domains of three PCPs that impart substantially different chain length distributions to surface polysaccharides. Despite very low sequence similarities, they have a common protomer structure with a long central α-helix extending 100 Å into the periplasm. The protomers self-assemble into bell-shaped oligomers of variable sizes, with a large internal cavity. Electron microscopy shows that one of the full-length PCPs has a similar organization as that observed in the crystal for its periplasmic domain alone. Functional studies suggest that the top of the PCP oligomers is an important region for determining polysaccharide modal length. These structures provide a detailed view of components of the bacterial polysaccharide assembly machinery.

A different conformation for EGC stator subcomplex in solution and in the assembled yeast V-ATPase: possible implications for regulatory disassembly.

Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy. In solution, EG forms a slightly kinked rod, which assembles with subunit C into an L-shaped structure. This model is supported by the microscopy data, which show three copies of EG with two of these linked by subunit C. However, the relative arrangement of the EG and C subunits in solution is more open than that in the holoenzyme, suggesting a conformational change of EGC during regulatory assembly and disassembly.

Highly sensitive and rapid radioimmunoassay for aldosterone in plasma and cell culture medium.

A radioimmunoassay for aldosterone was developed using a sensitive and specific antibody and 125Iodinated-aldosterone. This assay could be used for direct determination of aldosterone in cell culture medium or after extraction of aldosterone from plasma by solid phase procedure using C18 Sep-pak cartridges. The very low cross-reactivity of the antibody with cortisol and corticosterone (0.005% and 0.04% respectively) would allow the direct determination of aldosterone in cell culture medium without any prior extraction step. Since the incubation is performed at room temperature for 1 h and then, at 4 degrees C for 15 min, the results can be obtained in less than 3 h. The assay was linear from 0.5 fmol/tube to 1500 fmol/tube with an ED50 at 30 fmol/tube. The accuracy of the assay estimated using spiked plasma samples with a known amount of aldosterone give a coefficient of correlation of 0.97 (n = 10) between the aldosterone concentrations found and expected levels. The within-assay variability for plasma aldosterone varied from 4.7 to 11.1% and the between-assay variability ranged from 13.9 to 14.2%. The coefficient of correlation between plasma aldosterone measured by this new assay or by a current assay was 0.8 (n = 43). In summary, the combination of a shortened incubation time with a simple solid phase extraction for aldosterone in serum samples represents the major advantage of the present assay over the current methodology which usually requires a chromatographic separation of the mineralocorticoid prior to radioimmunoassay. Therefore this assay would be useful in experimental studies as well as in clinical laboratory.

An automated system for delivery of an unstable transcription factor to hematopoietic stem cell cultures

An automated delivery system for cell culture applications would permit studying more complex culture strategies and simplify measures taken to expose cells to unstable molecules. We are interested in understanding how intracellular TAT‐HOXB4 protein concentration affects hematopoietic stem cell (HSC) fate; however, current manual dosing strategies of this unstable protein are labor intensive and produce wide concentration ranges which may not promote optimal growth. In this study we describe a programmable automated delivery system that was designed to integrate into a clinically relevant, single‐use, closed‐system bioprocess and facilitate transcription factor delivery studies. The development of a reporter cell assay allowed for kinetic studies to determine the intracellular (1.4 ± 0.2 h) and extracellular (3.7 ± 1.8 h and 78 ± 27 h at 37°C and 4°C, respectively) half‐lives of TAT‐HOXB4 activity. These kinetic parameters were incorporated into a mathematical model, which was used to predict the dynamic intracellular concentration of TAT‐HOXB4 and optimize the delivery of the protein. The automated system was validated for primary cell culture using human peripheral blood patient samples. Significant expansion of human primitive progenitor cells was obtained upon addition of TAT‐HOXB4 without user intervention. The delivery system is thus capable of being used as a clinically relevant tool for the exploration and optimization of temporally sensitive stem cell culture systems. Biotechnol. Bioeng. 2009;103: 402–412.

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand‐binding domains of AMPA receptor subunits carry two conserved N‐glycosylation sites. In order to gain insight into the functional role of the corresponding N‐glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand‐binding characteristics, structural stability, cell‐surface expression, and channel properties of homomeric GluR‐D (GluR4) receptor and its soluble ligand‐binding domain (S1S2). GluR‐D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand‐binding properties were observed between the ‘wild‐type’ S1S2 and non‐glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N‐glycosylation. Purified glycosylated and non‐glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full‐length homomeric GluR‐D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild‐type GluR‐D. In outside‐out patches, GluR‐D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand‐binding domain glycans do not play any major role in receptor–ligand interactions, do not impart a stabilizing effect on the ligand‐binding domain, and are not critical for the formation and surface localization of homomeric GluR‐D AMPA receptors in HEK293 cells.

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines.

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R1A and ANF-R1C subtypes. A10 and NIH cells which express high density of ANF-R2 receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R1A and ANF-R1C subtypes are modulated in the same manner. In the presence of Mn2+, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg2+ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R1A, the ANF-R1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R1A subtype.

C-type natriuretic peptide in bovine chromaffin cells. The regulation of its biosynthesis and secretion.

We report here the regulation of the biosynthesis and the secretion of C‐type natriuretic peptide (CNP) in cultured bovine chromaffin cells. The combined treatment with protein kinase A and ‐C activators induced a 6‐fold increase of intracellular levels of CNP‐(1–103). The biosynthesized CNP‐(1–103) was co‐released with its mature forms, typically CNP‐(51–103), upon stimulation by nicotine or depolarizing agents. This confirms the neuropeptidic character of this third member of the natriuretic peptide family, which might act as a neuromodulator or neurotransmitter.

Crystallization and preliminary analysis of chondroitinase AC from Flavobacterium heparinum

Chondroitinase AC (E.C. 4.2.2.5) overexpressed in its host, Flavobacterium heparinum, was crystallized by vapor diffusion using polyethylene glycol methyl ether as precipitant. It crystallizes in the space group P43212 or its enantiomorph with a = b = 87.1 and c = 193.1 A and one molecule in the asymmetric unit. Crystals diffract to a maximum of 2.5 A resolution on a rotating-anode source. Screening for heavy-atom derivatives identified a lead compound that binds to a single site on the protein. Further screening is in progress.

Identification of the atrial natriuretic factor-RIC receptor subtype (B-clone) in cultured rat aortic smooth muscle cells

The present report demonstrates the presence in cultured rat aortic smooth muscle cells of a natriuretic factor receptor subtype with a specificity typical of the ANF‐RIC (B‐clone) receptor subtype. To prove the existence of this receptor subtype in this cell line we show that pCNP‐(82–103) is the most potent activator of the intrinsic guanylate cyclase activity, and that [125I]pCNP‐(82–103) binds to a specific receptor subtype which is insensitive to the ANF‐R2 specific ligand, C‐ANF. The investigation of its binding characteristics show the rank potency order of the natriuretic factors in competing for pCNP binding to be pCNP > pBNP > rANF. Furthermore it was possible to covalently photolable this receptor subtype with underivatized [125I]pCNP and show that it is composed of a single subunit of 130 kDa with very high specificity for pCNP.

Three-dimensional crystallization of membrane proteins.

Although the examination of the protein data bank reveals an important backlog in the number of three-dimensional structures of membrane proteins, several recent successes are serving as preludes to what will become a very prosperous decade in this field. Systematic investigations of various factors affecting the stability of membrane proteins, as well as their potential to crystallize three dimensionally, have paved the way for such achievements. The importance of the role of detergents both at the level of purification and crystallization is now well established. In addition, the recognition of the protein-detergent complex as the entity to crystallize, as well as the understanding of its physical-chemical properties and discovery of factors affecting these, have permitted the design of better crystallization strategies. As a consequence of the various efforts in the field, new crystallization methods for membrane proteins are being implemented. These have already been successful and are expected to contribute significantly to the future successes. This chapter will review some basic principles in membrane protein crystallization and give an overview of the current state of the art in the field. Some practical guidelines to help the novice approach the problem of membrane protein crystallization from the initial step of protein purification to crystallogenesis will also be given.