Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches.

BACKGROUND: The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy. METHODS: Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed. RESULTS: Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis. CONCLUSIONS: These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

Structure of the catalytic core of S. cerevisiae DNA polymerase eta: implications for translesion DNA synthesis.

DNA polymerase eta is unique among eukaryotic polymerases in its proficient ability to replicate through a variety of distorting DNA lesions. We report here the crystal structure of the catalytic core of S. cerevisiae DNA polymerase eta, determined at 2.25A resolution. The structure reveals a novel polydactyl right hand-shaped molecule with a unique polymerase-associated domain. We identify the catalytic residues and show that the fingers and thumb domains are unusually small and stubby. In particular, the unexpected absence of helices "O" and "O1" in the fingers domain suggests that openness of the active site is the critical feature which enables DNA polymerase eta to replicate through DNA lesions such as a UV-induced cis-syn thymine-thymine dimer.

Gene repression by coactivator repulsion.

We show that the IRF-2 oncoprotein represses virus-induced IFN-beta gene transcription via a novel mechanism. Virus infection induces recruitment of IRF-2 to some of the endogenous IFN-beta enhancers as part of the enhanceosome. Enhanceosomes bearing IRF-2 cannot activate transcription, due to the presence of a domain in IRF-2 that prevents enhanceosome-dependent recruitment of the CBP-Pol II holoenzyme complex. As a consequence, IRF-2 incorporation into enhanceosomes restricts the number of IFN-beta promoters directing transcription. Remarkably, deletion of the IRF-2 gene increases IFN-beta expression by expanding the number of cells capable of inducing IFN-beta gene transcription in response to virus infection.

Monomeric midkine induces tumor cell proliferation in the absence of cell-surface proteoglycan binding.

Midkine (MK), a retinoic acid-inducible heparin-binding protein, is a mitogen which initiates a cascade of intracellular protein tyrosine phosphorylation mediated by the JAK/STAT pathway after binding to its high affinity p200(+)/MKR cell surface receptor in the G401 cell line [Ratovitski, E. A. (1998) J. Biol. Chem. 273, 3654-3660]. In this study, we determined the biophysical characteristics of purified recombinant murine MK and analyzed the requirements for ligand multimerization and cell surface proteoglycan binding for the G401 cell mitogenic activity of MK. Our studies indicate that the secreted form of MK (M = 13 kDa) exists in solution as an asymmetric monomer with a frictional coefficient of 1. 48 and a Stokes radius of 23.7 A. By constructing bead models of MK using the program AtoB and the program HYDRO to predict the hydrodynamic properties of each model, our data suggest that MK has a dumb-bell shape in solution composed of independent N- and C-terminal domains separated by an extended linker. This asymmetric MK monomer is a biologically active ligand with mitogenic activity on G401 cells in vitro. Neither heparin-induced formation of noncovalent MK multimers nor tissue transglutaminase II covalent multimerization of MK enhanced MK mitogenic activity in this system. Since neither heparin competition nor cell treatment with chondroitinase ABC or heparinase III abolished the mitogenic effects of MK on G401 cells, cell-surface proteoglycan binding by MK does not appear to be a requirement for its observed mitogenic effects. These results provide strong evidence that the MK-specific p200(+)/MKR has distinctive biochemical properties which distinguish it from the receptor tyrosine phosphatase cell-surface proteoglycan PTPzeta/RPTPbeta and support the hypothesis that the diverse biological effects of MK are mediated by multiple cell-specific signal transduction receptors.

Crystallization and characterization of Pumilo: a novel RNA binding protein.

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6(3) with unit cell dimensions of a = b = 94.5 A, c = 228.9 A, alpha = beta = 90 degrees, gamma = 120 degrees and diffract to 2.6 A with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of alpha-helical secondary structure.

Structure of IRF-1 with bound DNA reveals determinants of interferon regulation.

The family of interferon regulatory factor (IRF) transcription factors is important in the regulation of interferons in response to infection by virus and in the regulation of interferon-inducible genes. The IRF family is characterized by a unique 'tryptophan cluster' DNA-binding region. Here we report the crystal structure of the IRF-1 region bound to the natural positive regulatory domain I (PRD I) DNA element from the interferon-beta promoter. The structure provides the first three-dimensional view of a member of the growing IRF family, revealing a new helix-turn-helix motif that latches onto DNA through three of the five conserved tryptophans. The motif selects a short GAAA core sequence through an obliquely angled recognition helix, with an accompanying bending of the DNA axis in the direction of the protein. Together, these features suggest a basis for the occurrence of GAAA repeats within IRF response elements and provide clues to the assembly of the higher-order interferon-beta enhancesome.

Expression, purification, and co-crystallization of IRF-I bound to the interferon-beta element PRDI.

Interferon regulatory factor 1 (IRF-1) is an essential factor involved in the regulation of type I interferon (IFN) and IFN-inducible genes. The protein consists of 329 amino acids that are highly conserved from mouse to human. Similar to other transcription factors, the protein is modular in nature with a basic N-terminal region involved in DNA binding and an acidic C-terminal region required for activation. We report here the expression, purification and co-crystallization of the minimal N-terminal region of IRF-1 involved in DNA binding (amino acids 1-113) with a 13 bp DNA fragment from the IFN-beta promoter. The crystals diffract to at least 3.0 A in resolution and belong to space group R3 with unit cell parameters of a = b = 84.8 A, c = 203.7 A.