Pulldown of human serum proteins with Trypanosoma brucei Invariant Surface Glycoprotein 75
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Blood Serum
SUBMITTER: 
Qi Wu
LAB HEAD: Christian Brix Folsted Andersen
PROVIDER: PXD047614 | Pride | 2024-02-06
REPOSITORIES: Pride
Dataset's files
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Project description:Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 alleles. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving allelic differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12, providing further evidence for hierarchical VSG expression, a conclusion that was confirmed by the novel application of diversity analysis to VSG expression. Our results indicate that long read analysis is a reliable tool for resolving diverse allele expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and identifying mosaic gene formation unprecedentedly early during the infection process.
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Project description:The human N-terminal acetyltransferase E (NatE) including its associated NatA co-translationally acetylates the N-terminus of about 40-60% of the proteome to mediate diverse biological processes including protein half-life, localization and protein interaction. In eukaryotes, the NatE complex contains the NAA50 catalytic subunit with substrate specificity for N-terminal methionine acetylation and NatA, which facilitates ribosomal targeting of the complex for co-translational activity. NatA, contains NAA10 catalytic and NAA15 auxiliary subunits, and forms a complex with a protein with intrinsic NAA10 inhibitory activity, HYPK. The molecular basis for how the human NAA10 and NAA50 catalytic subunits within NatE complex coordinate function and how HYPK regulates NatE activity is unknown. Here, we characterize the biochemical interplay between the human NAA10 and NAA50 catalytic subunits of NatE and its regulation by HYPK and correlate this to the cryo-EM structures of the human NatE and NatE/HYPK complexes. We show that NAA50 and HYPK exhibit negative cooperative binding to NatA in vitro and in human cells, by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contributes to NAA10 activity inhibition through structural alteration of the NAA10 substrate binding site. NatE is about 8-fold more active than NAA50, likely due to a reduced entropic cost for substrate binding through NatA tethering, but is inhibited by HYPK through structural alteration of the NatE substrate binding site. Taken together, these studies reveal the molecular basis for coordinated N-terminal acetylation by the NAA10 and NAA50 catalytic subunits of NatE and its modulation by HYPK.
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Project description:The surface proteins of parasitic protozoa mediate functions essential to survival within a host, including nutrient accumulation, environmental sensing and immune evasion. Several receptors involved in nutrient uptake and defence from the innate immune response have been described in African trypanosomes and, together with antigenic variation, contribute towards persistence within vertebrate hosts. Significantly, a superfamily of invariant surface glycoproteins (ISGs) populates the trypanosome surface, one of which, ISG75, is implicated in uptake of the century-old drug suramin. By CRISPR/Cas9 knockout and biophysical analysis, we show here that ISG75 directly binds suramin and mediates uptake of additional naphthol-related compounds, making ISG75 a conduit for entry of at least one structural class of trypanocidal compounds. However, ISG75 null cells present only modest attenuation of suramin sensitivity, have unaltered viability in vivo and in vitro and no alteration to suramin-invoked proteome responses. While ISG75 is demonstrated as a valid suramin cell entry pathway, we suggest the presence of additional mechanisms for suramin accumulation, further demonstrating the complexity of trypanosomatid drug interactions and potential for evolution of resistance.
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Project description:Casein is a common source of dietary protein in defined diets used to investigate the effect of diet on the gut microbiota. Previously it has been shown that the metagenomes of mice fed with a diet containing casein were contaminated with DNA from the bacterium Lactococcus lactis and that washing the casein with ethanol removed this contamination (https://doi.org/10.1016/j.chom.2019.06.013). We have identified the same phenomenon when identifying proteins from mouse diets with casein However, in contrast to DNA sequencing we did not see a reduction in L. lactis contamination in proteomes. Two lots each of standard casein and ethanol-washed casein were analyzed using proteomics.
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Project description:Trypanosoma brucei parasites successfully evade the host immune system by periodically switching the dense coat of Variant Surface Glycoproteins (VSG) at the cell surface. Each parasite expresses VSGs in a monoallelic fashion that is tightly regulated. The consequences of exposing multiple VSGs during an infection in terms of antibody response and disease severity remain unknown. In this study, we overexpressed a high mobility group box protein, TDP1, which was sufficient to open the chromatin of silent VSG expression sites, to disrupt VSG monoallelic expression and to generate viable and healthy parasites with a mixed VSG coat. Mice infected with these parasites mounted a multi-VSG antibody response, which rapidly reduced parasitemia. Consequently, we observed a prolonged mice survival in which nearly 90% of the mice survived a 30-day period infection with undetectable parasitemia. Immunodeficient RAG2 knock-out mice were unable to control the infection with TDP1-overexpressing parasites, showing that the adaptive immune response is critical to reduce disease severity. This study shows that simultaneous exposure of multiple VSGs is highly detrimental for the parasite even at the very early stages of infection, suggesting that drugs that disrupt VSG monoallelic expression could be used to treat trypanosomiasis.
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