Background: Parasite fatty acid synthesis is an attractive drug target but complex and poorly understood. Results: We delineate the molecular activity of two pathways in Toxoplasma combining metabolomic and genetic analyses.
Conclusion:The apicoplast is a significant source of fatty acids, and its products are further modified in the parasite endoplasmic reticulum. Significance: We define the metabolic host-parasite relationship with molecular resolution in intracellular parasites.
Background: Glycans are synthesized in the Golgi by sequentially acting glycosyltransferases, but it is not known how their functions are coordinated in live cells. Results: N-and O-glycosyltransferases form enzymatically active homo-and/or heteromeric complexes. Conclusion: Glycosyltransferases function as physically distinct enzyme complexes rather than single enzymes. Significance: The results help understand the overall functioning of the Golgi glycosylation pathways both in health and disease.
SUMMARY
Most simian immunodeficiency viruses use their Nef protein to antagonize the host restriction factor tetherin. A deletion in human tetherin confers Nef resistance, representing a hurdle to successful zoonotic transmission. HIV-1 group M evolved to utilize the viral protein U (Vpu) to counteract tetherin. Although HIV-1 group O has spread epidemically in humans, it has not evolved a Vpu-based tetherin antagonism. Here we show that HIV-1 group O Nef targets a region adjacent to this deletion to inhibit transport of human tetherin to the cell surface, enhances virion release, and increases viral resistance to inhibition by interferon-α. The Nef protein of the inferred common ancestor of group O viruses is also active against human tetherin. Thus, Nef-mediated antagonism of human tetherin evolved prior to the spread of HIV-1 group O and likely facilitated secondary virus transmission. Our results may explain the epidemic spread of HIV-1 group O.
IMPORTANCEIn this paper, we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses furthermore identify the tetraspanin protein family as a previously unrecognized target of Nef and Vpu activity. These findings have implications for the interpretation of effects detected for these accessory gene products on individual host cell receptors and illustrate the coevolution of Nef and Vpu function.
N-linked glycosylation is an essential posttranslational modification of proteins in eukaryotes. The substrate of N-linked glycosylation, dolichol pyrophosphate (DolPP)-GlcNAc(2)Man(9)Glc(3), is assembled through a complex series of ordered reactions requiring the translocation of the intermediate DolPP-GlcNAc(2)Man(5) structure across the endoplasmic-reticulum membrane. A young patient diagnosed with a congenital disorder of glycosylation characterized by an intracellular accumulation of DolPP-GlcNAc(2)Man(5) was found to carry a homozygous point mutation in the RFT1 gene. The c.199C-->T mutation introduced the amino acid substitution p.R67C. The human RFT1 protein shares 22% identity with its yeast ortholog, which is involved in the translocation of DolPP-GlcNAc(2)Man(5) from the cytosolic into the lumenal side of the endoplasmic reticulum. Despite the low sequence similarity between the yeast and the human RFT1 proteins, we demonstrated both their functional orthology and the pathologic effect of the human p.R67C mutation by complementation assay in Deltarft1 yeast cells. The causality of the RFT1 p.R67C mutation was further established by restoration of normal glycosylation profiles in patient-derived fibroblasts after lentiviral expression of a normal RFT1 cDNA. The definition of the RFT1 defect establishes the functional conservation of the DolPP-GlcNAc(2)Man(5) translocation process in eukaryotes. RFT1 deficiency in both yeast and human cells leads to the accumulation of incomplete DolPP-GlcNAc(2)Man(5) and to a profound glycosylation disorder in humans.
Most organelles within the exocytic and endocytic pathways typically acidify their interiors, a phenomenon that is known to be crucial for their optimal functioning in eukaryotic cells. This review highlights recent advances in our understanding of how Golgi acidity is maintained and regulated, and how its misregulation contributes to organelle dysfunction and disease. Both its biosynthetic products (glycans) and protein-sorting events are highly sensitive to changes in Golgi luminal pH and are affected in certain human disease states such as cancers and cutis laxa. Other potential disease states that are caused by, or are associated with, Golgi pH misregulation will also be discussed.
3-ketoacyl-CoA synthase catalyses the initial condensation reaction during fatty acid elongation using malonyl-CoA and long-chain acyl-CoA as substrates. Previously, it was reported that several genes encoding putative cotton 3-ketoacyl-CoA synthases were significantly up-regulated during early cotton fibre development. In this study, GhCER6 cDNA that contains an open reading frame of 1479 bp, encoding a protein of 492 amino acid residues homologous to the Arabidopsis condensing enzyme CER6, was isolated and cloned. In situ hybridization results demonstrated that GhCER6 mRNA was detected only in the elongating wild-type cotton fibre cells. When GhCER6 was transformed to the Saccharomyces cerevisiae elo3 deletion mutation strain that was deficient in the production of 26-carbon fatty acids and displayed a very slow-growth phenotype, the mutant cells were found to divide similarly compared with those of the wild-type cells. Further, heterologous expression of GhCER6 restored the viability of the S. cerevisiae haploid elo2 and elo3 double-deletion strain. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that GhCER6 was enzymatically active since the yeast elo2 and elo3 double-deletion mutant expressing the cotton gene produced very-long-chain fatty acids that are essential for cell growth. The results suggest that GhCER6 encodes a functional 3-ketoacyl-CoA synthase.
The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection.
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