Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes

In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron microscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.     

Biotechnological approaches for L-ascorbic acid production

Over the past decade there has been increasing pressure to develop alternatives to the Reichstein process, a largely chemical synthesis by which the vast majority of world vitamin C (L-ascorbic acid, L-AA) is produced. The pressures include increasing environmental concerns and legislation, and the need to increase process efficiency and reduce capital costs. The development of efficient fermentation processes in the past ten years has also represented a catalyst for change. Here, we describe the development of biotechnological alternatives for the synthesis of Reichstein intermediates by industrial microorganisms. The recent elucidation of the plant biosynthetic pathway represents new opportunities not only for the direct synthesis of L-AA by fermentation but also for the production of human crop plants and animal fodder with enhanced nutritional value. We discuss the potential for these developments in the light of recent findings concerning L-AA biosynthesis in plants.     

Participation of CaMKII in neuronal plasticity and memory formation

1. The unique biochemical properties of Ca²⁺/calmodulin (CaM)-dependent protein kinase II have made this enzyme one of the paradigmatic models of the forever searched memory molecule.2. In particular, the central participation of CaMKII as a sensor of the Ca²⁺ signals generated by activation of NMDA receptors after the induction of long-term plastic changes, has encouraged the use of pharmacological, genetic, biochemical, and imaging tools to unveil the role of this kinase in the acquisition, consolidation, and expression of different types of memories.3. Here we review some of the more exciting discoveries related to the mechanisms involved in CaMKII activation and synaptic plasticity.     

Structural features and conformational equilibria of 310-helical peptides in solution by spectroscopic and molecular mechanics studies

The structural features and conformational equilibria of a series of short, linear Calpha-methylvaline [(alphaMe)Val]-based peptides in methanol were investigated by combining fluorescence resonance energy transfer measurements and molecular mechanics data. IR spectra were employed to determine their secondary structure, which exhibits an intramolecularly H-bonded, 3(10)-helix conformation that is affected by backbone distortions that are enhanced by the shortness of the main chain.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Presenilin-1 exists in both pre- and post-Golgi compartments and recycles via COPI-coated membranes

Presenilin-1 is involved in intramembrane proteolysis of various proteins, but its intracellular site of action has remained elusive. Here, we determined by quantitative immunogold-electron microscopy that presenilin-1 in Chinese hamster ovary cells is present in pre-Golgi compartments as well as at the plasma membrane and endosomes. Notably, a high percentage of presenilin-1 resides in COPI-coated membranes between the endoplasmic reticulum and the Golgi complex, indicating significant recycling to the endoplasmic reticulum. By contrast, the inactive aspartate mutant presenilin-1_D257A is relatively excluded from COPI-coated membranes, concomitant with increased post-Golgi levels. These data provide critical evidence for the scenario that the complex containing presenilin-1 can serve as γ-secretase at the plasma membrane or endosomes and suggest a role for COPI-mediated retrograde transport in regulating post-Golgi levels of presenilin-1.     

Upregulation of CYP2e1 and CYP3a activities in histamine-deficient histidine decarboxylase gene targeted mice

Histamine is a biogenic amine with multiple physiological functions. Its importance in allergic inflammation is well characterized; moreover, it plays a role in the regulation of gastric acid production, various hypothalamic functions, such as food uptake, and enhancing TH2 balance during immune responses. Using histidine decarboxylase gene targeted (HDC(-/-)) BALB/c mice, we studied the effect of the absence of histamine on four cytochrome p450 enzyme activities. Their selective substrates were measured: ethoxyresorufin O-dealkylase activity of CYP1A, pentoxyresorufin O-dealkylase activity of CYP2B, chlorzoxazone 6-hydroxylase activity of CYP2E1 and ethylmorphine N-demethylase activity of CYP3A.The results indicate a significant elevation of CYP2E1 and CYP3A activities, however, no change in CYP1A and CYP2B activities was seen in HDC targeted mice compared to wild type controls with identical genetic backgrounds.     

A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-?Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]? [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.     

Emerin expression at the early stages of myogenic differentiation

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.