ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family

In human cells, Ero1-Lalpha and -Lbeta (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein--protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1-Lalpha. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport-competent Ig-K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short-lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1-Lalpha redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.     

Dendritic cell immunogenicity is regulated by peroxisome proliferator-activated receptor gamma

Dendritic cells (DC) are the most potent APCs known that play a key role for the initiation of immune responses. Ag presentation to T lymphocytes is likely a constitutive function of DC that continues during the steady state. This raises the question of which mechanism(s) determines whether the final outcome of Ag presentation will be induction of immunity or of tolerance. In this regard, the mechanisms controlling DC immunogenicity still remain largely uncharacterized. In this paper we report that the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma), which has anti-inflammatory properties, redirects DC toward a less stimulatory mode. We show that activation of PPAR-gamma during DC differentiation profoundly affects the expression of costimulatory molecules and of the DC hallmarker CD1a. PPAR-gamma activation in DC resulted in a reduced capacity to activate lymphocyte proliferation and to prime Ag-specific CTL responses. This effect might depend on the decreased expression of costimulatory molecules and on the impaired cytokine secretion, but not on increased IL-10 production, because this was reduced by PPAR-gamma activators. Moreover, activation of PPAR-gamma in DC inhibited the expression of EBI1 ligand chemokine and CCR7, both playing a pivotal role for DC migration to the lymph nodes. These effects were accompanied by down-regulation of LPS-induced nuclear localized RelB protein, which was shown to be important for DC differentiation and function. Our results suggest a novel regulatory pathway for DC function that could contribute to the regulated balance between immunity induction and self-tolerance maintenance.     

Geometrical isomerism of monounsaturated fatty acids: thiyl radical catalysis and influence of antioxidant vitamins

Thiyl radicals generated either from thiols or disulfides act as the catalyst for the cis-trans isomerization of a variety of monounsaturated fatty acid methyl esters in homogeneous solution. Similar results have also been obtained using alpha-lipoic acid and its reduced form. The effectiveness of the isomerization processes in the presence of the most common antioxidants has been addressed. The ability of thiyl radical scavenging was found to increase along the series alpha-tocopherol < ascorbic acid < all-trans retinol. The cis-trans isomerization of fatty acid residues in multilamellar vesicles of dioleoyl phosphatidyl choline by thiyl radical, in the absence and presence of the various antioxidants, has also been studied in detail. The influence of the isomerization process on the phospholipid bilayer has been tested by permeability measurements of vesicles and it is clearly shown that trans fatty acid-containing membranes have intermediate properties between those formed by all-cis and saturated components. This study contributes to the understanding of radical processes that can alter or protect the naturally occurring cis geometry of unsaturated lipids in cell membranes and demonstrates a new role of essential antioxidants.     

A genomic overview of pyridoxal-phosphate-dependent enzymes

Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free-living prokaryotes, almost 1.5% of all genes code for PLP-dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP-dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP-dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP-dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP-dependent activities than it has genes for PLP-dependent enzymes. This observation presumably applies to many other classes of protein-encoding genes.     

PBX1 nuclear export is regulated independently of PBX-MEINOX interaction by PKA phosphorylation of the PBC-B domain

The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. We investigated the processes controlling PBX1 nuclear export. Here we show that in the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. Our results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain.     

Conformational constraints of tyrosine in protein tyrosine kinase substrates: Information about preferred bioactive side-chain orientation

The side-chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149-152), was fixed in the gauche (+) or gauche (-) conformation by using the 7-hydroxy-1,2,3,4-tetrahydro isoquinoline-3-carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine-type (Hba) structure. The proposed side-chain orientations were confirmed by the analysis of the (1)H-NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This "rotamer scan" of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src-related tyrosine kinases Lyn and c-Fgr.     

MISSING FLOWERS gene controls axillary meristems initiation in sunflower

The initiation and growth of axillary meristems are fundamental components of plant architecture. Here, we describe the mutant missing flowers (mf) of Helianthus annuus characterized by the lack of axillary shoots. Decapitation experiments and histological analysis indicate that this phenotype is the result of a defect in axillary meristem initiation. In addition to shoot branching, mutation affects floral differentiation. The indeterminate inflorescence of sunflower (capitulum) is formed of a large flat meristem which produces floret primordia in multiple spirals. In wildtype plants a bisecting crease divides each primordium in two distinct bumps that adopt different fate. The peripheral (abaxial) part of the primordium becomes a small leaf-like bract and the adaxial part becomes a flower. In the mf mutant, the formation of flowers at the axil of bracts is precluded. Histological analyses show that in floret primordia of the mutant a clear subdivision in dyads is not established. The primordia progressively bend inside and only large involucral floral bracts are developed. The results suggest that the MISSING FLOWERS gene is essential to provide or perceive an appropriate signal to the initiation of axillary meristems during both vegetative and reproductive phases.     

Salicylate biosynthesis: overexpression, purification, and characterization of Irp9, a bifunctional salicylate synthase from Yersinia enterocolitica

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.     

Inbreeding patterns in La Cabrera, Spain: dispensations, multiple consanguinity analysis, and isonymy

Marital structure and inbreeding coefficients were analyzed in La Cabrera, an isolated mountain region in northwestern Spain. A total of 5,714 marriages were celebrated from 1880 to 1989 in the 37 parishes of the area. The total frequency of consanguineous marriages (up to the fourth degree) is 23.05%; multiple consanguineous marriages are remarkably common, reaching 5.43% of the total. The first cousin/second cousin ratio (referred to as kinship-type frequencies) is 0.43. The inbreeding values are the highest recorded in Spain and in Europe: alpha3 is 4.82 x 10(-3) for the whole period and alpha4 is 6.78 x 10(-3) for 1880--1919. The temporal trend of inbreeding shows high values (alpha3 > 4.5 x 10(-3)) for a particularly long period (1900--1959) and a rapid decline from 1960 onward. This historical inbreeding trend is clearly related to changes in population size. The frequencies of multiple consanguineous marriages and the analysis of isonymy show that the inbreeding structure is related to geographic and demographic factors. Comparing the results at two hierarchical levels (La Cabrera as a whole and the 37 parishes individually), we conclude that the inbreeding values are affected by internal geographic subdivision of the population (Wahlund effect). Social and cultural factors, such as avoidance of or preference for consanguineous marriages, are less important but depend on the kinship type involved.     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.