Carboxyl terminus structural requirements for glycosyl-phosphatidylinositol anchor addition to cell surface proteins.

Glycosyl phosphatidylinositol (GPI)-anchored proteins contain in their COOH-terminal region a peptide segment that is thought to direct glycolipid addition. This signal has been shown to require a pair of small amino acids positioned 10-12 residues upstream of an hydrophobic C-terminal domain. We analysed the contribution of the region separating the anchor acceptor site and the C-terminal hydrophobic segment by introducing amino acid deletions and substitutions in the spacer element of the GPI-anchored Thy-1 glycoprotein. Deletions of 7 amino acids in this region, as well as the introduction of 2 charged residues, prevented the glycolipid addition to Thy-1, suggesting that the length and the primary sequence of the spacer domain are important determinants in the signal directing GPI anchor transfer onto a newly synthesized polypeptide. Furthermore, we tested these rules by creating a truncated form of the normally transmembranous Herpes simplex virus I glycoprotein D (gDI) and demonstrating that when its C-terminal region displays all the features of a GPI-anchored protein, it is able to direct glycolipid addition onto another cell surface molecule.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

The functional and biological properties of whey proteins : prospects for the development of functional foods

Selostus: Heraproteiinit terveysvaikutteisten elintarvikkeiden kehittämisessä

Rpe65 microarrays, from genes to phosphorylated proteins

Purpose:To describe a novel in silico method to gather and analyze data from high-throughput heterogeneous experimental procedures, i.e. gene and protein expression arrays. Methods:Each microarray is assigned to a database which handles common data (names, symbols, antibody codes, probe IDs, etc.). Links between informations are automatically generated from knowledge obtained in freely accessible databases (NCBI, Swissprot, etc). Requests can be made from any point of entry and the displayed result is fully customizable. Results:The initial database has been loaded with two sets of data: a first set of data originating from an Affymetrix-based retinal profiling performed in an RPE65 knock-out mouse model of Leber's congenital amaurosis. A second set of data generated from a Kinexus microarray experiment done on the retinas from the same mouse model has been added. Queries display wild type versus knock out expressions at several time points for both genes and proteins. Conclusions:This freely accessible database allows for easy consultation of data and facilitates data mining by integrating experimental data and biological pathways.

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation.

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly modify in the three groups, novel structural components were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m3G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hi-bernating dormice, were present in much higher amounts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs.

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.

Audit report on the Iowa Petroleum Underground Storage Tank Board (UST Board) for the year ended June 30, 2010

Audit report on the Iowa Petroleum Underground Storage Tank Board (UST Board) for the year ended June 30, 2010

The antiviral adaptor proteins Cardif and Trif are processed and inactivated by caspases

The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses

Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17.

The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

Patterns of calcium-binding proteins in human inferior colliculus: identification of subdivisions and evidence for putative parallel systems.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures.

Identifying knots in proteins.

Polypeptide chains form open knots in many proteins. How these knotted proteins fold and finding the evolutionary advantage provided by these knots are among some of the key questions currently being studied in the protein folding field. The detection and identification of protein knots are substantial challenges. Different methods and many variations of them have been employed, but they can give different results for the same protein. In the present article, we review the various knot identification algorithms and compare their relative strengths when applied to the study of knots in proteins. We show that the statistical approach based on the uniform closure method is advantageous in comparison with other methods used to characterize protein knots.

Controlled expression of recombinant proteins in Physcomitrella patens by a conditional heat-shock promoter: a tool for plant research and biotechnology.

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using beta-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 degrees C. In contrast, a short non-damaging heat-treatment at 38 degrees C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 degrees C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.

The emerging importance of the SPX domain-containing proteins in phosphate homeostasis

Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.

Differential phosphoproteomics of fragmenting yeast vacuoles

Many organelles exist in an equilibrium of fragmentation into smaller units and fusion into larger structures, which is coordinated with cell division, the increase in cell mass, and envi¬ronmental conditions. In yeast cells, organelle homeostasis can be studied using the yeast vacuole (lysosome) as a model system. Yeast vacuoles are the main compartment for degrada¬tion of cellular proteins and storage of nutrients, ions and metabolites. Fission and fusion of vacuoles can be induced by hyper- and hypotonic shock in vivo, respectively, and have also been reconstituted in vitro using isolated vacuoles. The conserved serine/threonine kinase TOR (target of rapamycin) is a central nutrient sensor and regulates cell growth and metabolism. In yeast, there are two TOR proteins, Torlp and Tor2p, which are part of larger protein complexes, TORCI and TORC2. Only TORCI is rapamycin-sensitive. Disregulation of TOR signaling is linked to a multitude of diseases in humans, e.g. cancer, neurodegenerative diseases and metabolic syndrome. It has been shown that TORCI localizes to the vacuole membrane, and recent findings of our laboratory demonstrated that TORCI positively regulates vacuole fragmentation. This suggests that the fragmentation machinery should contain target proteins phosphorylated by TORCI. I explored the rapamycin-and fission-dependent vacuolar phosphoproteome during frag¬mentation, using a label-free mass-spectrometry approach. I identified many vacuolar factors whose phosphorylation was downregulated in a TORCI- and fission-dependent manner. Among them were known protein complexes that are functionally linked to fission or fusion, like the HOPS, VTC and FAB1 complexes. Hence, TORCI-dependent phosphorylations might positively regulate vacuole fission. Several candidates were chosen for detailed microscopic analysis of in vivo vacuole frag-mentation, using deletion mutants. I was able to identify novel factors not previously linked to fission phenotypes, e.g. the SEA complex, Pib2, and several vacuolar amino acid transporters. Transport of neutral and basic amino acids across the membrane seems to control vacuole fission, possibly via TORCI. I analyzed vacuolar fluxes of amino acids in wildtype yeast cells and found evidence for a selective vacuolar export of basic amino acids upon hyperosmotic stress. This leads me to propose a model where vacuolar export of amino acids is necessary to reshape the organelle under salt stress. - Le nombre et la taille de certaines organelles peut être déterminé par un équilibre entre la fragmentation qui produit des unités plus petites et la fusion qui génère des structures plus larges. Cet équilibre est coordonné avec la division cellulaire, l'augmentation de la masse cellulaire, et les conditions environnementales. Dans des cellules de levure, l'homéostasie des organelles peut être étudié à l'aide d'un système modèle, la vacuole de levure (lysosome). Les vacuoles constituent le principal compartiment de la dégradation des protéines et de stockage des nutriments, des ions et des métabolites. La fragmentation et la fusion des vacuoles peuvent être respectivement induites par un traitement hyper- ou hypo-tonique dans les cellules vivantes. Ces processus ont également été reconstitués in vitro en utilisant des vacuoles isolées. La sérine/thréonine kinase conservée TOR (target of rapamycin/cible de la rapamycine) est un senseur de nutriments majeur qui régule la croissance cellulaire et le métabolisme. Chez la levure, il existe deux protéines TOR, Torlp et Tor2p, qui sont les constituants de plus grands complexes de protéines, TORCI et TORC2. TORCI est spécifiquement inhibé par la rapamycine. Une dysrégulation de la signalisation de TOR est liée à une multitude de maladies chez l'homme comme le cancer, les maladies neurodégénératives et le syndrome métabolique. Il a été montré que TORCI se localise à la membrane vacuolaire et les découvertes récentes de notre laboratoire ont montré que TORCI régule positivement la fragmentation de la vacuole. Ceci suggère que le mécanisme de fragmentation doit être contrôlé par la phosphorylation de certaines protéines cibles de TORCI. J'ai exploré le phosphoprotéome vacuolaire lors de la fragmentation, en présence ou absence de rapamycine et dans des conditions provoquant la fragmentation des organelles. La méthode choisie pour réaliser la première partie de ce projet a été la spectrométrie de masse différentielle sans marquage. J'ai ainsi identifié plusieurs facteurs vacuolaires dont la phosphorylation est régulée d'une manière dépendante de TORCI et de la fragmentation. Parmi ces facteurs, des complexes protéiques connus qui sont fonctionnellement liées à fragmentation ou la fusion, comme les complexes HOPS, VTC et FAB1 ont été mis en évidence. Par conséquent, la phosphorylation dépendante de TORCI peut réguler positivement la fragmentation des vacuoles. Plusieurs candidats ont été choisis pour une analyse microscopique détaillée de la fragmentation vacuolaire in vivo en utilisant des mutants de délétion. J'ai été en mesure d'identifier de nouveaux facteurs qui n'avaient pas été encore associés à des phénotypes de fragmentation tels que les complexes SEA, Pib2p, ainsi que plusieurs transporteurs vacuolaires d'acides aminés. Le transport des acides aminés à travers la membrane semble contrôler la fragmentation de la vacuole. Puisque ces transporteurs sont phosphorylés par TORCI, ces résultats semblent confirmer la