Project description:DPANN are a widespread and highly diverse group of archaea characterised by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. DPANNs are predominantly obligate ectosymbionts that depend on their host for their survival and proliferation. Despite the recent expansion in this clade, the structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we used electron cryotomography (cryo-ET) to reveal that the Candidatus Micrarchaeota” (ARM-1) interacts with its host through intercellular proteinaceous nanotubes. These tubes (~4.5 nm wide) originate in the host, extend all the way to the DPANN cytoplasm and act like tunnels for intercellular exchange. Combining cryo-ET and sub-tomogram averaging, we revealed the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states, providing mechanistic insights into substrate exchange. Additionally, we performed comparative proteomics and genomic analyses to identify host proteomic changes in response to the DPANN attachment. Our results showed striking alterations in host-proteome during symbiosis and upregulation/downregulation of key cellular pathways. Collectively, these results provided unprecedented insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.
Project description:The haematopoietic cytokine thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for maintenance of the haematopoetic stem cell compartment. Tpo is a heavily glycosylated, hepatocyte-derived cytokine which functions by binding to its receptor (TpoR) on target cells and thereby activating intracellular signalling cascades that induce their proliferation and/or differentiation. In addition to its role in signal propagation, TpoR is expressed on the surface of platelets, where it contributes to regulation of Tpo levels by sequestering circulating cytokine. TpoR belongs to the homodimeric Class I cytokine receptor family but is unusual due to a duplication of the Cytokine binding Homology Region (CHR). Almost thirty years after initial discovery of TpoR, the structure of the human Tpo:TpoR interaction was recently reported. Here we determine the structure of extracellular portion of the murine Tpo:TpoR signalling complex using single particle cryo-EM. The structure reveals that Tpo:TpoR forms a largely symmetrical 1:2 complex. The cytokine cross-links the same site on the membrane-distal CHR of both receptor chains using opposing surfaces and with significantly different affinities. This orients the two membrane-proximal CHRs such that they contact one another adjacent to the plasma membrane. The potential cytokine-binding site in CHR2 is glycosylated and does not interact with Tpo. A large insertion in CHR1 that is unique to Tpo forms a partially structured loop that is disulphide bonded to CHR2 and, in one receptor chain, contacts cytokine. Biochemical analyses indicate that the glycosylated C-terminal domain of Tpo does not influence receptor binding. We demonstrate that the therapeutic TpoR agonist Romiplostim binds to the same site on the receptor as does cytokine. Our study characterises the Tpo/TpoR interaction structurally and biochemically to allow for the future development of potent TpoR agonists for therapeutic use.
Project description:Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown, in part due to the lack of tools with which to study the glycan. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN and EndoNDM on streptavidin-coated magnetic or agarose beads. We showed that immobilizing EndoN improves enzyme usefulness by allowing for effective removal of the enzyme from samples, while retaining hydrolase activity. Additionally, immobilization of EndoNDM allowed for the enrichment of polysialylated proteins from complex mixtures for their identification via mass spectrometry. We identified QSOX2 as a novel polysialylated protein secreted from MCF-7 cells. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.
Project description:Comparative proteomics of Bacteroides thetaiotaomicron samples comparing the total membrane (TM) and outer membrane vesicles (OMV) of WT B. thetaiotaomicron and delta 4364