Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the human placenta, mononuclear cytotrophoblasts in a human chorionic gonadotropin (hCG)-driven process fuse to form multinucleated syncytia that allow the exchange of nutrients and gases between the maternal and fetal circulation. Experiments in which protein kinase A (PKA) is displaced from A-kinase anchoring proteins (AKAPs), or in which specific AKAPs are depleted by siRNA-mediated knockdown, point to ezrin as a scaffold required for hCG-, cAMP-and PKA-mediated regulation of the fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we show that ezrin directs PKA to a molecular complex of connexin 43 (Cx43, also known as GJA1) and zona occludens-1 (ZO-1, also known as TJP1). A combination of knockdown experiments and reconstitution with ezrin or Cx43 with or without the ability to bind to its interaction partner or to PKA demonstrate that ezrin-mediated coordination of the localization of PKA and Cx43 is necessary for discrete control of Cx43 phosphorylation and hCG-stimulated gap junction communication that triggers cell fusion in cytotrophoblasts.

The microheterogeneity of recombinant human phenylalanine hydroxylase (hPAH) was investigated by isoelectric focusing and 2D electrophoresis. When expressed in Escherichia coli four main components (denoted hPAH I-IV) of < 50 kDa were observed on long-term induction at 28±37 8C with isopropyl thio-b-d-galactoside (IPTG), differing in pI by about 0.1 pH unit. A similar type of microheterogeneity was observed when the enzyme was expressed (1 h at 37 8C) in an in vitro transcription-translation system, including both its nonphosphorylated and phosphorylated forms which were separated on the basis of a difference in mobility on SDS/ PAGE. Experimental evidence is presented that the microheterogeneity is the result of nonenzymatic deamidations of labile amide containing amino acids. When expressed in E. coli at 28 8C, the percentage of the acidic forms of the enzyme subunit increased as a function of the induction time with IPTG, representing about 50% on 8 h induction. When the enzyme obtained after 2 h induction (containing mainly hPAH I) was incubated in vitro, its conversion to the acidic components (hPAH II±IV) revealed a pH and temperature dependence characteristic of a nonenzymatic deamidation of asparagine residues in proteins, with the release of ammonia. Comparing the microheterogeneity of the wild-type and a truncated form of the enzyme expressed in E. coli, it is concluded that the labile amide groups are located in the catalytic domain as defined by crystal structure analysis [Erlandsen, H., Fusetti, F., Martõ Ânez, A., Hough, E., Flatmark, T. & Stevens, R. C. (1997) Nat. Struct. Biol. 4, 995±1000]. It is further demonstrated that the progressive deamidations which occur in E. coli results in a threefold increase in the catalytic efficiency (V max /[S] 0.5 ) of the enzyme and an increased susceptibility to limited tryptic proteolysis, characteristic of a partly activated enzyme. The results also suggest that deamidation may play a role in the long term regulation of the catalytic activity and the cellular turnover of this enzyme.

Human CD4 ؉ FoxP3 ؉ T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4 ؉ CD25 ؉ and CD4 ؉ CD25 ؊ T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells

The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (L-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMPdependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available threedimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 Å (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.