The Gup1 homologue of Trypanosoma brucei is a GPI glycosylphosphatidylinositol remodelase

Glycosylphosphatidylinositol (GPI) lipids of Trypanosoma brucei undergo lipid remodelling, whereby longer fatty acids on the glycerol are replaced by myristate (C14:0). A similar process occurs on GPI proteins of Saccharomyces cerevisiae where Per1p first deacylates, Gup1p subsequently reacylates the anchor lipid, thus replacing a shorter fatty acid by C26:0. Heterologous expression of the GUP1 homologue of T. brucei in gup1Delta yeast cells partially normalizes the gup1Delta phenotype and restores the transfer of labelled fatty acids from Coenzyme A to lyso-GPI proteins in a newly developed microsomal assay. In this assay, the Gup1p from T. brucei (tbGup1p) strongly prefers C14:0 and C12:0 over C16:0 and C18:0, whereas yeast Gup1p strongly prefers C16:0 and C18:0. This acyl specificity of tbGup1p closely matches the reported specificity of the reacylation of free lyso-GPI lipids in microsomes of T. brucei. Depletion of tbGup1p in trypanosomes by RNAi drastically reduces the rate of myristate incorporation into the sn-2 position of lyso-GPI lipids. Thus, tbGup1p is involved in the addition of myristate to sn-2 during GPI remodelling in T. brucei and can account for the fatty acid specificity of this process. tbGup1p can act on GPI proteins as well as on GPI lipids.     

Progression of Liver Fibrosis in HIV/HCV Co-Infection: A Comparison between Non-Invasive Assessment Methods and Liver Biopsy

Objectives

To evaluate the diagnostic performance of seven non-invasive tests (NITs) of liver fibrosis and to assess fibrosis progression over time in HIV/HCV co-infected patients.

Methods

Transient elastography (TE) and six blood tests were compared to histopathological fibrosis stage (METAVIR). Participants were followed over three years with NITs at yearly intervals.

Results

Area under the receiver operating characteristic curve (AUROC) for significant fibrosis (> = F2) in 105 participants was highest for TE (0.85), followed by FIB-4 (0.77), ELF-Test (0.77), APRI (0.76), Fibrotest (0.75), hyaluronic acid (0.70), and Hepascore (0.68). AUROC for cirrhosis (F4) was 0.97 for TE followed by FIB-4 (0.91), APRI (0.89), Fibrotest (0.84), Hepascore (0.82), ELF-Test (0.82), and hyaluronic acid (0.79). A three year follow-up was completed by 87 participants, all on antiretroviral therapy and in 20 patients who completed HCV treatment (9 with sustained virologic response). TE, APRI and Fibrotest did not significantly change during follow-up. There was weak evidence for an increase of FIB-4 (mean increase: 0.22, p = 0.07). 42 participants had a second liver biopsy: Among 38 participants with F0-F3 at baseline, 10 were progessors (1-stage increase in fibrosis, 8 participants; 2-stage, 1; 3-stage, 1). Among progressors, mean increase in TE was 3.35 kPa, in APRI 0.36, and in FIB-4 0.75. Fibrotest results did not change over 3 years.

Conclusion

TE was the best NIT for liver fibrosis staging in HIV/HCV co-infected patients. APRI-Score, FIB-4 Index, Fibrotest, and ELF-Test were less reliable. Routinely available APRI and FIB-4 performed as good as more expensive tests. NITs did not change significantly during a follow-up of three years, suggesting slow liver disease progression in a majority of HIV/HCV co-infected persons on antiretroviral therapy.     

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.     

Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei , PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAi against ethanolamine-phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk-like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock-down of ET. Interference with phospholipid synthesis also impaired normal cell-cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline-/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non-division of the nucleus and production of zoids.