Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA - Crystal structure of an oligomeric complex of yeast SmF

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.

Cytokine-related genes identified from the RIKEN full-length mouse cDNA data set

To identify novel cytokine-related genes, we searched the set of 60,770 annotated RIKEN mouse cDNA clones (FANTOM2 clones), using keywords such as cytokine itself or cytokine names (such as interferon, interleukin, epidermal growth factor, fibroblast growth factor, and transforming growth factor). This search produced 108 known cytokines and cytokine-related products such as cytokine receptors, cytokine-associated genes, or their products (enhancers, accessory proteins, cytokine-induced genes). We found 15 clusters of FANTOM2 clones that are candidates for novel cytokine-related genes. These encoded products with strong sequence similarity to guanylate-binding protein (GBP-5), interleukin-1 receptor-associated kinase 2 (IRAK-2), interleukin 20 receptor alpha isoform 3, a member of the interferon-inducible proteins of the Ifi 200 cluster, four members of the membrane-associated family 1-8 of interferon-inducible proteins, one p27-like protein, and a hypothetical protein containing a Toll/Interleukin receptor domain. All four clones representing novel candidates of gene products from the family contain a novel highly conserved cross-species domain. Clones similar to growth factor-related products included transforming growth factor beta-inducible early growth response protein 2 (TIEG-2), TGFbeta-induced factor 2, integrin beta-like 1, latent TGF-binding protein 4S, and FGF receptor 4B. We performed a detailed sequence analysis of the candidate novel genes to elucidate their likely functional properties.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Polarization vision and its role in biological signaling

Visual pigments, the molecules in photoreceptors that initiate the process of vision, are inherently dichroic, differentially absorbing light according to its axis of polarization. Many animals have taken advantage of this property to build receptor systems capable of analyzing the polarization of incoming light, as polarized light is abundant in natural scenes (commonly being produced by scattering or reflection). Such polarization sensitivity has long been associated with behavioral tasks like orientation or navigation. However, only recently have we become aware that it can be incorporated into a high-level visual perception akin to color vision, permitting segmentation of a viewed scene into regions that differ in their polarization. By analogy to color vision, we call this capacity polarization vision. It is apparently used for tasks like those that color vision specializes in: contrast enhancement, camouflage breaking, object recognition, and signal detection and discrimination. While color is very useful in terrestrial or shallow-water environments, it is an unreliable cue deeper in water due to the spectral modification of light as it travels through water of various depths or of varying optical quality. Here, polarization vision has special utility and consequently has evolved in numerous marine species, as well as at least one terrestrial animal. In this review, we consider recent findings concerning polarization vision and its significance in biological signaling.

Overlapping motifs in the membrane-proximal region of cytokine receptor accessory and signaling subunits

The membrane-proximal cytoplasmic region of cytokine receptors (CRs) is highly conserved and essential for receptor activation. In particular this region is essential for the activation of members of the Janus family of protein kinases (JAK) which results in initiation of receptor signaling. We have examined the sequence of this region in a number of CR signaling and accessory subunits with a view to better delineating motifs that play an important role in initiating receptor activity. Here, we have delineated two distinct proline-rich motifs in the membrane-proximal domains of cytokine receptors. Their configuration and distribution among CR subunits strongly suggest a model in which the two motifs act in a concerted manner to induce full receptor and JAK activation. (C) 2004 Elsevier Ltd. All rights reserved.

Downregulation of ultraspiracle gene expression delays pupal development in honeybees

Ecdysteroids regulate many aspects of insect physiology after binding to a heterodimer composed of the nuclear hormone receptor proteins ecdysone receptor (EcR) and ultraspiracle (Use). Several lines of evidence have suggested that the latter also plays important roles in mediating the action of juvenile hormone (JH) and, thus, integrates signaling by the two morphogenetic hormones. By using an RNAi approach, we show here that Us p participates in the mechanism that regulates the progression of pupal development in Apis mellifera, as indicated by the observed pupal developmental delay in usp knocked-down bees. Knock-down experiments also suggest that the expression of regulatory genes such as ftz transcription factor 1 (ftz-f1) and juvenile hormone esterase (jhe) depend on Usp. Vitellogenin (vg), the gene coding the main yolk protein in honeybees, does not seem to be under Usp regulation, thus suggesting that the previously observed induction of vg expression by JH during the last stages of pupal development is mediated by yet unknown transcription factor complexes. (C) 2008 Elsevier Ltd. All rights reserved.

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 ( neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between yield and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially screen, in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts. (C) 2004 Wiley Periodicals, Inc.

Incorporation of antigenic GPI-proteins from Leishmania amazonensis to membrane mimetic systems: Influence of DPPC/cholesterol ratio

The reconstitution of membrane proteins into liposomes is a useful tool to prepare antigenic components that induce immunity. We have investigated the influence of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol molar ratio on the incorporation of a GPI-protein from Leishmania amazonensis on liposomes and Langmuir monolayers. The latter system is a well behaved and practical model, for understanding the effect of variables such as surface composition and lipid packing on protein incorporation. We have found that the DPPC/cholesterol molar ratio significantly alters the incorporation of the GPI-protein. In the absence of cholesterol, reconstitution is more difficult and proteoliposomes cannot be prepared, which we correlated with disruption of the DPPC layer. Our results provide important information that Could be employed in the development of a vaccine system for this disease or be used to produce other GPI-systems for biotechnological application. (c) 2009 Elsevier Inc. All rights reserved.

Homocysteine, Alzheimer genes and proteins, and measures of cognition and depression in older men

The epsilon4 allele of apolipoprotem E (APOE), and the plasma levels of APOE, amyloid beta-protein precursor, arnyloid beta1-40 (Abeta40) and homocysteine, (Hcy) have all been correlated with the presence of dementia. Mutations in the methylnetetrahydrofolate reductase enzyme (MTHFR) have been associated with elevated levels of Hcy. This study explored the association of these factors with cognition and depression in community dwelling older men. Two hundred and ninety-nine men, mean age 78.9 years (SD 2.8), were studied in this cross-sectional survey. Mean plasma Hcy was 13.5 (SD 5.3) mumol/L. The MTHFR genotype had no obvious impact on Hey levels. Ln Hcy and Ln Abeta40 were both inversely correlated with calculated glomerular filtration rate (cGFR), r = -0.41 (p < 0.001) and r = -0.28 (p < 0.001), respectively. There was a positive correlation between Ln Hey and Ln Abeta40, r = 0.19 (p < 0.001), which remained significant after adjusting for cGFR, with a doubling of Hcy associated with a 24% increase of Abeta40. The e4 allele was associated with increased depressive symptoms as measured by the Geriatric Depression Scale-15, Odds ratio (OR) = 2.59 (95% CI 1.06-6.34) and poorer performance on the Clock Drawing Test, OR = 2.32 (95% CI: 1.25-4.29). There was a positive association between Abeta40 and Hcy, even after adjustment for cGFR in this sample of well, community dwelling older men. This association may help elucidate the link between elevated levels of Hey and Alzheimer's disease.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.