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Title: The role of p24 proteins in the ER to Golgi transport of Toll-like receptors
Author: Holm, Julia Elvira Johanna
ISNI:       0000 0004 7961 9593
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognizing conserved structural features of bacterial or viral origin and regulate the initiation of the innate immune response. TLR signaling is subjected to a dynamic and complex regulation that remains poorly understood. Our research suggests that TMED7 (Transmembrane Emp24 Domain-7) and TMED2, which are members of the p24 (TMED) family of small type I transmembrane receptors that operate in the early secretory pathway, are involved in the ER to Golgi transport of TLRs. Protein interaction studies performed in HEK293T cells by Proximity Ligand Assay (PLA) reveal that TMED7 interacts with TLR2 and TLR4, but not with TLR3. In contrast, TMED2 interacts with TLR2, TLR4 and TLR3. Different TLRs were co-expressed with ER-export deficient TMED7 and TMED2 and their cellular localisation observed using confocal microscopy. These data suggest that TMED7 is required for ER-export of TLR4 and TLR2 but not TLR3, while TMED2 is required for the ER-export of TLR2, TLR3 and TLR4. Here, evidence is also presented showing that the N-glycosylation state affects the level of ER export of TLR2. Together, these findings suggest that TMED2 and TMED7 associate with some TLRs and cooperates to facilitate their transport from the ER to the Golgi. In addition, CRISPR-Cas9 knock-out of TMED2 and TMED10 in HEK293T cells resulted in poor cell viability implying that some members of the p24 family are important for normal cellular functions. To investigate the oligomeric properties of TMED7, recombinant TMED7 ectodomain was produced in HEK293T cells and purified protein was subjected to analysis by Analytical Ultra Centrifugation (AUC). These data suggest that TMED7 ectodomain is predominantly monomeric but higher order associations are possible.
Supervisor: Gay, Nicholas John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: p24 proteins ; TMED7 ; TMED2 ; TMED10 ; Toll-like receptors ; TLR4 ; TLR2 ; TLR3 ; Microscopy ; CRISPR-CAS9 ; ER ; Golgi ; secretory pathway ; co-localisation ; proximity ligand assay ; macrophages ; HEK293T cells