2007: Arroyo Javier; Sarfati Jaqueline; Baixench Marie Thérése; Ragni Enrico; Guillén Marivi; Rodriguez-Peña José Manuel; Popolo Laura; Latgé Jean Paul
The GPI-anchored Gas and Crh families are fungal antigens
Yeast (Chichester, England) 2007;24(4):289-96.
The cell wall is the first interface between a fungus and its extracellular environment.
Glycosyltransferases involved in the formation and dynamic remodelling of the polysaccharide network of the cell wall have recently been identified.
The best characterized ones belong to the
Gas family, which elongates beta(1,3)-glucans, and to the
Crh family, which are involved in the cross-linking of chitin to beta(1,6)-glucan. (SEE BELOW ON THE LARGE)
All these proteins carry a glycosylphosphatidylinositol (GPI) anchor. (SEE BELOW ON THE LARGE)
In this work, we show that recombinant soluble forms of Gas1-5 and Crh1p from Saccharomyces cerevisiae and their orthologous proteins Gel1-Gel2 and Crf1 from Aspergillus fumigatus are specifically recognized by antibodies present in the sera of patients with Aspergillus or Candida infections.
Quantification of the antibody titres against recombinant Gas/Gel and Crh/Crf proteins separated aspergilloma and candidiasis patients from non-infected individuals.
Cross-reactivity was seen between the antibody response of patients with aspergillosis and candidiasis towards the Gas/Gel and Crh/Crf proteins.
These results suggest that GPI-anchored cross-linking enzymes are relevant immunologically reactive constituents of the cell wall that may play a role during human fungal infections.
http://www.biomedexperts.com/Abstract.bme/17397107/The_GPI-anchored_Gas_and_Crh_families_are_fungal_antigens
The Corticotropin-Releasing Hormone (CRH) family of peptides
The 41-amino acid sequence of CRH was first discovered in sheep by Vale et al. in 1981
http://en.wikipedia.org/wiki/Corticotropin-releasing_hormone
CRH is a 41-amino acid peptide that was first isolated from the sheep hypothalamus.
It has since been found in a large variety of species with a high degree of sequence identity, emphasizing the importance of this hormone.
The human CRH gene consists of two exons separated by an intron in its 5' untranslated region. The rat and ovine CRH genes have the same organization. While CRH accounts for a large part of the corticotropin-releasing activity of extracts in the hypothalamus, it does not account for all of it.
In fact there appears to be a wider family featuring structurally related peptides, including sauvagine in amphibia, urotensin I in teleost fish and urocortin in mammals.
Urocortin is localizaed in the rat midbrain region, testis, cardiac myocytes, thymus, spleen, kidney and human reproductive tissues.
CRH-like activity is widely distributed in:
-central nervous system,
-adrenals,
-lungs,
-liver,
-stomach,
-pancreas,
-small intestine and
-reproductive tissues and in a variety of
-human tumours.
The following systems are affected by CRH through extrapituitary mechanisms:
-cardiovascular regulation,
-respiration,
-appetite control,
-glucose metabolism,
-immune function, and
-cognitive and motor behaviour.
Moreover, immunoreactive CRH found in reproductive tissues can exert a number of effects. It can participate in intrauterine inflammatory processes of early pregnancy.
Recently, two new members of the CRH neuropeptide family have been cloned:
-stress-copin-related peptide (SRP)/urocortin II and
-stresscopin (SCP)/urocotrtin III.
SRP/ urocortin III mRNA expression have been detected in the gastrointestinal tract, muscle, adrenal gland and skin.
CRH Receptors
CRH Receptors belong to the class II superfamily of "brain-gut" neuropeptide receptors, which all contain 7 transmembrane helical domanins and share considerable sequence identity with one another.
Currently there are 2 known classes of CRH receptors, termed type 1 and type 2, that have been cloned from a number of vertebrate species and are encoded by separate genes.
CRH-R1 shares 70% identity with CRH-R2 and both receptors are present in structurally distinct isoforms.
The CRH-R1 gene expresses multiple subtypes, 1alpha-h, which are produced by differential exon splicing.
Each CRH-R1 variant has a defect in its expression, binding or signalling characteristics; for example, CRH-R1 beta contains a 29-amino-acid insertion in its first intracellular loop, allowing only weak coupling to the stimulatory G-protein.
The CRH-R2 gene expresses 3 known subtypes, 2alpha, 2beta and 2epsilon, that are produced by the use of alternative 5" exons and hence differ only at the N-terminus, which forms part of the first extracellular domain.
CRH-R2 mRNA is mainly expressed in peripheral tissue, particularly in cardiac myocytes, lung, skeletal muscles, ovary and gastrointestinal tract.
In the brain, the highest densities are in the parvovetricular nucleus of the hypothalamus, the amygdala and the lateral septum.
More recently, in the diploid catfish species, a third CRH receptor (CRH-R3), has been identified. This novel CRH receptor is structurally closer to cat-fish CRH-R1 than CRh-R2 and binds with a 5-fold higher affinity than urotensin I abd sauvagne. CRH-R3 in the catfish is expressed in the pituitary gland, urophysis and brain.
This multiplicity is receptor subtypes and ligands provides for diversity of receptor expression and signalling.
CRH receptor sensitivity
Mammalian CRH-R1 receptors have an equal, yet high affinity for CRH, urocortin, sauvagne and urotensin I, while not showing any affinity for urocortin II or III.
In contrast, CRH-R2 receptors show a clear preference in their affinity for urocortin-like ligands.
Urocortins I, II and III, all show high-affinity binding to CRH-R2, while mammalian CRH binds weakly.
CRH receptors are highly promiscuous. Not only do they bind a number of different ligands, but they can also activate different G-alpha subunits. Due to the complex nature of mammalian cells, the precise details of the coupling of the CRH receptors to their cognate G-protein are unknown.
LAB YEAST-CRH EXPERIENCE:
To investigate such coupling, we have utilized a YEAST reporter strain in which the pheromone-response pathways were adapted to allow ligand-dependent signalling of heterologously G-proteins or or YEAST-mammalian chimaeric G-alpha proteins.
We expressed one representative of each family of CRH receptors (CRH-R1 alpha and CRH-R2 beta) within SCHIZOSACCHAROMYCES pombe cells.
(Schizosaccharomyces pombe, also called "fission yeast", is a species of yeast. It is used as a model organism in molecular and cell biology.) - wikipedia)
http://en.wikipedia.org/wiki/Schizosaccharomyces_pombe
Due to modifications of the cell, the CRH receptor subtypes were the only GPCRs present within the cell.
When the cells were challenged with exogenous CRH, we observed functional coupling and signalling of the CRH-R1 alpha receptor via Gas and Gai.
In contrast, urocortin I activated Gas and Gaq.
Urocortins II and III did not activate any G-proteins. Using cells transfected with CRH-R2b, we confirmed that urocortins II and III signal via this receptor subtype.
These results are as expected, since CRH has a high affinity for CRH-R1 receptors,
but only a low affinity for type 2 receptors.
Contained within this study, we investigated a number of different ligands, and made changes to
components within the Sz. pombe cells to allow a more accurate evaluation of CRH signalling.
From our results it is clear that CRH-receptor±G-protein coupling}signalling is far from simple,
and may occur in a ligand-dependent manner.
The understanding of the precise nature of CRH signalling for both receptor families and each receptor subtype is of paramount importance in understanding stress responses.
Intracellular signalling molecules
In many tissues (e.g. brain, heart, myometrium), stimulation of either type of CRH receptor by CRH or CRH-related peptides leads to the activation of adenylate cyclase and increased cAMP levels.
However, in certain tissues (i.e. testes, placenta), CRH is unable to activate the adenylate cyclase pathway, whereas it can stimulate alternative signalling cascades, such as stimulation of phosphoinositol hydrolysis, but with reduced efficacy.
As mentioned above, several studies in native tissues and artificial expression systems have demonstrated multiple G-protein activation, a finding that predicts activation of several different second messenger signals and suggests that CRH and CRH-related peptides can generate various responses in different target tissues.
Indeed, it has been shown thus far that the CRH receptors can modulate various intacellular signals, such as protein kinase.
Protein kinase, nitric oxide synthase, guanylate cyclase, prostaglandins, steroidogenic enzymes, FasL production and apoptosis.
The CRH-receptor±MAPK interaction appears to be of particular interest, since in some
cellular systems (myometrial or HEK cells overexpressing CRH receptors), urocortin or
sauvagine, but not CRH, can activate the p42}p44 MAPK system [47,48].
This effect appears to be mediated primarily, but not exclusively, via activation of the Gq±InsP$±PKC pathway.
Studies on G-protein activation and second messenger
production demonstrated that urocortin was significantly more potent than CRH in activating the Gq±InsP$±PKC pathway.
This shows the central role of the agonist±CRH-receptor±G-protein complex in determining activation of signalling cascades.
In contrast, in neuronal and hippocampal cells, both CRH and urocortin can activate the MAPK cascade, a signalling pathway that mediates the neuroprotective effects of these peptides
[49,50]; this appears to occur via activation of the Gs}adenylate cyclase system.
Without any doubt, many more studies are required to elucidate the signalling pathways that are influenced by the family of CRH peptides in different tissues.
SEE THE BOOK AT:
http://www.biochemsoctrans.org/bst/030/0428/0300428.pdf
Vol. 12, Issue 11, 3631-3643, November 2001
Signaling through Adenylyl Cyclase Is Essential for Hyphal Growth and Virulence in the Pathogenic Fungus Candida albicans
Cintia R. C. Rocha,* Klaus Schröppel, Doreen Harcus,* Anne Marcil,* Daniel Dignard,* Brad N. Taylor, David Y. Thomas,*§ Malcolm Whiteway,*§ and Ekkehard Leberer*¶
*Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada; the Institute of Clinical Microbiology, Immunology, and Hygiene, University of Erlangen, D-91054 Erlangen, Germany; and the Departments of Anatomy and Cell Biology, §Biology, and Experimental Medicine, McGill University, Montreal, Canada
The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals.
This morphogenetic switching has been implicated in the development of pathogenicity.
We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p.
It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast.
The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes.
C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans.
The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages.
However, morphogenetic switching was restored by exogenous cAMP.
On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p.
These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching.
Homozygous cacdc35 cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections.
These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.
http://www.molbiolcell.org/cgi/content/abstract/12/11/3631
Cyclic adenosine monophosphate (cAMP)
From Wikipedia, the free encyclopedia
Cyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate) is a second messenger important in many biological processes. cAMP is derived from adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway.
cAMP is synthesised from ATP by adenylyl cyclase located at the cell membranes.
cAMP and its associated kinases function in several biochemical processes, including the regulation of glycogen, sugar, and lipid metabolism.
Some research has suggested that a deregulation of cAMP pathways and an aberrant activation of cAMP-controlled genes is linked to the growth of some cancers.
http://en.wikipedia.org/wiki/Cyclic_adenosine_monophosphate
Copyright © 1997 Published by Elsevier Science B.V.
Glycosyl-phosphatidylinositol anchored membrane enzymes (GPI anchors)
Nigel M. Hooper,
School of Biochemistry and Molecular Biology, The University of Leeds, Leeds LS2 9JT, UK
Received 20 October 1996; accepted 23 July 1997. ; Available online 11 March 1999.
Abstract
Several mammalian enzymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol (GPI) structure.
These include acetylcholinesterase, alkaline phosphatase, membrane dipeptidase and 5′-nucleotidase.
All GPI anchors determined to date have the conserved core structure ethanolamine-PO4-6Manα1-2Manα1-6Manα1-4GlcNH2α1-6myo-inositol-1-PO−4 lipid.
In most mammalian GPI anchors the lipid is 1-alkyl-2-acyl-glycerol, although in porcine membrane dipeptidase it is diacylglycerol.
Attached to the conserved core are various side chain residues that appear to be either protein- or tissue-specific.
In addition to membrane attachment, a GPI anchor may confer additional properties on the protein, such as the susceptibility to cleavage by phospholipases and the potential to cluster in detergent-insoluble domains.
GPI anchors can also act as intracellular targeting signals, in transmembrane signalling, in the clathrin-independent endocytic process of potocytosis and as hormone mediators.
Thus, a GPI anchor can confer additional properties on enzymes that may be important in their physiological and pathophysiological functioning.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T57-3W0K17R-2&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1062001067&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=05e1d002f735043de9be6fd944c73467
Resistance to candidiasis and macrophage activity in chitin-treated mice
Aitor Rementeríaa, Fernando Abaituaa, Roberto García-Tobalinaa, Fernando Hernandoa, José Pontóna, María Jesús Sevillaa
a Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco, Apdo. 644, Bilbao, E-48080, Spain
The effect of chitin, a polysaccharide of the cell wall of Candida albicans, on both the survival of C. albicans infected mice and the activity of the murine peritoneal macrophages has been studied. Pretreatment of mice with 30 mg kg−1C. albicans chitin enhanced the survival of the infected animals. The protective effect was concomitant with an enhancement of both phagocytic and candidacidal activities of the peritoneal macrophages. Chitin by itself did not induce the nitric oxide (NO) synthase in the macrophages, which remained at a level similar to that shown by the macrophages from untreated animals. The administration of 10 mg kg−1C. albicans chitin diminished the long term survival of the infected animals. This effect was coincident with a lower candidacidal activity and NO production by the macrophages of the chitin treated and infected animals, compared to the untreated infected animals.
Received 15 May 1997, Revised 18 July 1997, Accepted 23 July 1997
http://www3.interscience.wiley.com/journal/119157751/abstract?CRETRY=1&SRETRY=0
Crh1p and Crh2p are required for the cross-linking of chitin to β(1-6)glucan in the Saccharomyces cerevisiae cell wall
Affiliation(s) du ou des auteurs / Author(s) Affiliation(s)(1) National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Biochemistry and Genetics, Bethesda, MD 20892, ETATS-UNIS(2) Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, ESPAGNE
Résumé / Abstract
In budding yeast, chitin is found in three locations:
-at the primary septum, largely in free form,
-at the mother-bud neck, partially linked to β(1-3)glucan,
-and in the lateral wall, attached in part to β(1-6)glucan.
By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to β(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant.
This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to β(1-6)glucan.
Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Δ and gas1Δ mutants, which are impaired in cell wall synthesis.
A temperature shift from 30°C to 38°C increased the proportion of chitin attached to β(1-6)glucan.
The expression of CRH1, but not that of CRH2, was also higher at 38°C in a manner dependent on the cell integrity pathway.
Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-β(1-6)glucan complex is found, was greatly enhanced at 38°C.
Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.
http://cat.inist.fr/?aModele=afficheN&cpsidt=18487525
