31 research outputs found
Isolation and Characterisation of the GPI:Protein Transamidase From Leishmania mexicana
Many eukaryotic cell surface proteins are attached to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. Trypanosomatid parasitic protozoa such as Leishmania and Trypanosoma brucei make extensive use of this method of protein surface attachment. The perceived importance of GPI-anchored proteins makes the GPI biosynthetic pathway a good target for anti-parasite chemotherapy. The terminal step in the pathway is the addition of complete pre-formed GPI anchors to the carboxyl-termini of proteins. This involves replacing a GPI signal sequence with a GPI anchor in a transamidation reaction. Two of the proteins involved in this step have been identified in yeast and mammals. One of these components, GPI8, has significant homology to a family of plant cysteine proteinases, the legumains, and GPI8 is therefore believed to be the catalytic subunit of the transamidase. The work described in this thesis focussed on the cloning of the GPI8 gene from Leishmania mexicana and its characterisation. The predicted protein shares 31% identity with yeast and human homologues. The nucleotide sequence of a fragment of the T. brucei GPI8 gene has also been obtained. Targeted gene replacement of the single copy L. mexicana GPI8 produced GPI8 null mutants. The loss of GPI8 was confirmed by Southern blotting. The phenotype of the GPI8 null mutants was analysed with the following findings: (1) Mutant promastigotes grow well in culture. (2) GP63 is not detected on the promastigote surface and is greatly reduced in promastigote lysates. (3) Episomal re-expression of GPI8 restored GP63 to the cell surface. (4) GPI8 null mutants are able to infect macrophages in vitro to approximately wild type levels, and replicate within macrophages. (5) GPI8 null mutants are capable of forming lesions in mice. These data show that GPI-anchored proteins of L. mexicana are not essential for growth of promastigotes, invasion of macrophages by promastigotes, or infection of mice. It remains to be established if GPI-anchored proteins are required for survival in the sandfly. Sequence comparison of L. mexicana, yeast and human GPI8 proteins identified two potential active site cysteine residues. Mutation in which Cys216 was converted to a glycine led to loss of GPI8 activity, as assessed by GP63 surface expression, indicating that this may be the active site cysteine. Recombinant GPI8 was produced in E. coli and used to inoculate rabbits for the production of anti-serum. These antibodies detected recombinant protein but failed to detect protein in L. mexicana cell lysates. Antibodies raised against a peptide of GPI8 also recognised recombinant GPI8, but not GPI8 in L. mexicana lysates. A protein of 38-40 kDa, however, was detected in cell lysates of T. brucei with the antirecombinant GPI8 antibodies. This antiserum gave a similar immunofluorescence pattern in T. brucei to those of an epitope-tagged ribosomal protein, QM. There is some overlap in fluorescence patterns between tagged QM and the endoplasmic reticulum marker protein BiP. This suggests that GPI8 is located in the ER of T. brucei, the same subcellular location that has been identified in yeast. A fusion protein of GPI8 and GFP was expressed in L. mexicana to attempt to localise GPI8 in this organism. The fusion protein, however, was unable to restore GPI8 function in the null mutant, therefore data on its localisation cannot be inferred
Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion
Artículo científico -- Universidad de Costa Rica, Instituto de Investigaciones en Salud. 2014Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenicmouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with amajor role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell divisionindependent mutational pathway.Universidad de Costa Rica. Instituto de Investigaciones en SaludInstitute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of
Glasgow, GlasgowParis Descartes-Sorbonne Paris Cité UniversityUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA
Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins
Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins
Chromosome and gene copy number variation allow major structural change between species and strains of **Leishmania**
Get Out of the Way! How a Simulation Changed How I Think About Teaching, Turned on My Students, and Ruined My Other Classes
A contingency framework of the relationship between the use of IT and organisational design variables
The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19 Delta fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19 Delta fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19 Delta fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.Publisher PDFPeer reviewe